lc ms ms analysis all peptide  (Thermo Fisher)


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    Structured Review

    Thermo Fisher lc ms ms analysis all peptide
    GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif <t>analysis</t> for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr <t>peptide</t> abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) <t>MS/MS</t> spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).
    Lc Ms Ms Analysis All Peptide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation"

    Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

    Journal: Journal of Proteome Research

    doi: 10.1021/acs.jproteome.8b00895

    GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif analysis for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr peptide abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) MS/MS spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).
    Figure Legend Snippet: GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif analysis for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr peptide abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) MS/MS spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).

    Techniques Used: Sequencing, Mass Spectrometry

    Data processing of product ion triggered MS/MS spectra. (A) A schematic of SEQUEST-HT searches of triggered EThcD and HCD spectra using the second Af1521 replicate of IFN-γ-treated THP-1 cells. (B) Number of peptide-spectrum matches (PSMs) of assigned ADPr and unmodified peptides from the triggered spectra. (C–E) Distribution of isolation interference for product ion triggered or DDA PSMs. (F) Number of ADPr peptides with high confidence detected by either EThcD or HCD. (G) Venn diagrams comparing ADPr peptide identifications between EThcD and HCD for all ADPr peptides, and those with > 95% ADPr acceptor site probability.
    Figure Legend Snippet: Data processing of product ion triggered MS/MS spectra. (A) A schematic of SEQUEST-HT searches of triggered EThcD and HCD spectra using the second Af1521 replicate of IFN-γ-treated THP-1 cells. (B) Number of peptide-spectrum matches (PSMs) of assigned ADPr and unmodified peptides from the triggered spectra. (C–E) Distribution of isolation interference for product ion triggered or DDA PSMs. (F) Number of ADPr peptides with high confidence detected by either EThcD or HCD. (G) Venn diagrams comparing ADPr peptide identifications between EThcD and HCD for all ADPr peptides, and those with > 95% ADPr acceptor site probability.

    Techniques Used: Mass Spectrometry, Isolation

    2) Product Images from "Separation and Identification of 1,2,4-Trihydroxynaphthalene-1-O-glucoside in Impatiens glandulifera Royle"

    Article Title: Separation and Identification of 1,2,4-Trihydroxynaphthalene-1-O-glucoside in Impatiens glandulifera Royle

    Journal: Molecules

    doi: 10.3390/molecules18078429

    Chromatographic profile of the Impatiens glandulifera Royle methanolic extract of the roots measured as total ion current (TIC) by LC-MS (APCI). MS spectrum of THNG is shown on the Figure 3 .
    Figure Legend Snippet: Chromatographic profile of the Impatiens glandulifera Royle methanolic extract of the roots measured as total ion current (TIC) by LC-MS (APCI). MS spectrum of THNG is shown on the Figure 3 .

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    3) Product Images from "Hsc70 Is a Novel Interactor of NF-kappaB p65 in Living Hippocampal Neurons"

    Article Title: Hsc70 Is a Novel Interactor of NF-kappaB p65 in Living Hippocampal Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065280

    Hsc70 is a novel neuronal interaction partner of NF-κB. A. Porcine brain extracts were immunoprecipitated with anti NF-κB p65 antibody or isotype control on protein G sepharose in presence of cross-linker. The IP were separated in a 1D SDS gel. Each lane (p65 precipitate and control) were cut into 36 slices and prepared for MS by trypsin digestion. All 36 slices were analyzed by MS. Seven samples in range of 95 to 60 and 27 to 24 kDa were additionally analyzed by LC-ESI-MS/MS. The hits identified by MS included the heat shock cognate Hsc70 as a potential interaction partner of NF-κB p65. B. HEK293 co-transfected with p65-flag and Hsc70-myc or IκBε-myc were lysed followed by co-immunoprecipitation in presence of cross-linker using αmyc (IP) antibody with subsequent WB analysis. A clear interaction band (WB: αFlag) was detectable if myc-tagged IκBε and flag-tagged NF-κB p65 were co-transfected. Similarly, co-transfection of p65-flag and Hsc70-myc resulted in a clear interaction band (WB: αFlag), whereas no band was observed in negative controls (no p65-flag, or no IκBε-myc or Hsc70-myc). Lysates were used as input control. C. Neuronal proteins influence the interaction of NF-κB p65 with Hsc70. IP (αmyc) was performed in presence of cross-linker (DSP) and/or brain lysates with subsequent analysis by western blot. Interaction bands (WB: αFlag) were detectable in cross-linked samples for myc-tagged IκBε and flag tagged NF-κB p65 as well as for Hsc70-myc and NF-κB p65-flag. Combination of cross-linker and brain lysates resulted in stronger interaction band (WB: αFlag) for Hsc70-myc and NF-κB p65-flag. Without cross-linker no interaction bands was detectable.
    Figure Legend Snippet: Hsc70 is a novel neuronal interaction partner of NF-κB. A. Porcine brain extracts were immunoprecipitated with anti NF-κB p65 antibody or isotype control on protein G sepharose in presence of cross-linker. The IP were separated in a 1D SDS gel. Each lane (p65 precipitate and control) were cut into 36 slices and prepared for MS by trypsin digestion. All 36 slices were analyzed by MS. Seven samples in range of 95 to 60 and 27 to 24 kDa were additionally analyzed by LC-ESI-MS/MS. The hits identified by MS included the heat shock cognate Hsc70 as a potential interaction partner of NF-κB p65. B. HEK293 co-transfected with p65-flag and Hsc70-myc or IκBε-myc were lysed followed by co-immunoprecipitation in presence of cross-linker using αmyc (IP) antibody with subsequent WB analysis. A clear interaction band (WB: αFlag) was detectable if myc-tagged IκBε and flag-tagged NF-κB p65 were co-transfected. Similarly, co-transfection of p65-flag and Hsc70-myc resulted in a clear interaction band (WB: αFlag), whereas no band was observed in negative controls (no p65-flag, or no IκBε-myc or Hsc70-myc). Lysates were used as input control. C. Neuronal proteins influence the interaction of NF-κB p65 with Hsc70. IP (αmyc) was performed in presence of cross-linker (DSP) and/or brain lysates with subsequent analysis by western blot. Interaction bands (WB: αFlag) were detectable in cross-linked samples for myc-tagged IκBε and flag tagged NF-κB p65 as well as for Hsc70-myc and NF-κB p65-flag. Combination of cross-linker and brain lysates resulted in stronger interaction band (WB: αFlag) for Hsc70-myc and NF-κB p65-flag. Without cross-linker no interaction bands was detectable.

    Techniques Used: Immunoprecipitation, SDS-Gel, Mass Spectrometry, Transfection, Western Blot, Cotransfection

    4) Product Images from "Interaction proteome of human Hippo signaling: modular control of the co-activator YAP1"

    Article Title: Interaction proteome of human Hippo signaling: modular control of the co-activator YAP1

    Journal: Molecular Systems Biology

    doi: 10.1002/msb.201304750

    A systematic affinity purification mass spectrometry ( AP ‐ MS ) approach to define the human Hpo pathway interaction proteome Selection of primary and secondary baits in this study. Baits were selected sequentially, starting with the core components of the Hpo kinase signaling pathway and extended based on obtained AP ‐ MS results or homology to D rosophila H po components. Biochemical workflow for native protein complex purification from HEK 293‐Flp T‐rex cells. Bait proteins were expressed from a tetracycline‐inducible CMV promoter, with a N‐terminal Strep‐ HA fusion tag following induction with doxycycline for 24 h. Cells were lysed, complexes affinity‐purified and processed for analysis by tandem mass spectrometry. Data analysis pipeline. Acquired mass spectra from 90 experiments (at least 2 biological replicates per bait) were searched with X!Tandem. Search results were statistically validated by the Trans‐Proteomic Pipeline ( TPP ) to match a protein identification false discovery rate of
    Figure Legend Snippet: A systematic affinity purification mass spectrometry ( AP ‐ MS ) approach to define the human Hpo pathway interaction proteome Selection of primary and secondary baits in this study. Baits were selected sequentially, starting with the core components of the Hpo kinase signaling pathway and extended based on obtained AP ‐ MS results or homology to D rosophila H po components. Biochemical workflow for native protein complex purification from HEK 293‐Flp T‐rex cells. Bait proteins were expressed from a tetracycline‐inducible CMV promoter, with a N‐terminal Strep‐ HA fusion tag following induction with doxycycline for 24 h. Cells were lysed, complexes affinity‐purified and processed for analysis by tandem mass spectrometry. Data analysis pipeline. Acquired mass spectra from 90 experiments (at least 2 biological replicates per bait) were searched with X!Tandem. Search results were statistically validated by the Trans‐Proteomic Pipeline ( TPP ) to match a protein identification false discovery rate of

    Techniques Used: Affinity Purification, Mass Spectrometry, Selection, Purification

    5) Product Images from "The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in ?-Granules Trafficking"

    Article Title: The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in ?-Granules Trafficking

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085833

    TLR4 interacts with myosin-9. (A) Identification of myosin-9 as a TLR4-interacting protein by co-IP and mass spectrometry. Washed platelet lysates were prepared for IP with mouse IgG- or anti-TLR4-conjugated agarose beads. The precipitated proteins were resolved by SDS-PAGE and revealed by Coomassie Blue staining. The stars indicated the protein bands that were pulled down with the anti-TLR4 antibody but not by mouse IgG. The stars indicated myosin-9 that was identified by nano-LC/MS/MS on an LCQ Deca XP Plus ion trap mass spectrometer.
    Figure Legend Snippet: TLR4 interacts with myosin-9. (A) Identification of myosin-9 as a TLR4-interacting protein by co-IP and mass spectrometry. Washed platelet lysates were prepared for IP with mouse IgG- or anti-TLR4-conjugated agarose beads. The precipitated proteins were resolved by SDS-PAGE and revealed by Coomassie Blue staining. The stars indicated the protein bands that were pulled down with the anti-TLR4 antibody but not by mouse IgG. The stars indicated myosin-9 that was identified by nano-LC/MS/MS on an LCQ Deca XP Plus ion trap mass spectrometer.

    Techniques Used: Co-Immunoprecipitation Assay, Mass Spectrometry, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy

    6) Product Images from "Label-Free Proteomics Assisted by Affinity Enrichment for Elucidating the Chemical Reactivity of the Liver Mitochondrial Proteome toward Adduction by the Lipid Electrophile 4-hydroxy-2-nonenal (HNE)"

    Article Title: Label-Free Proteomics Assisted by Affinity Enrichment for Elucidating the Chemical Reactivity of the Liver Mitochondrial Proteome toward Adduction by the Lipid Electrophile 4-hydroxy-2-nonenal (HNE)

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2016.00002

    Heatmap visualization of quantitative values of the 182 proteins enriched at the protein level . Left: heatmap of the 182 proteins identified and quantified over all six HNE exposure groups; Right: Zoomed-in region of the heatmap focusing on the most abundantly detected and identified putative HNE protein adducts. After affinity capture, samples were trypsin-digested and analyzed using LC-MS. Protein quantification was based on the peak intensity of the 3 most intense peptides. A total of 182 proteins were quantified. From top to bottom the 182 identified proteins are listed and each row represents a protein and its corresponding abundance. From left to right, HNE concentrations that were used for the in vitro exposure experiments of the mitochondrial protein samples. The color in each cell represents protein abundance obtained from the “Hi3” peptide intensity approach: red is more abundant and dark green is less abundant. Black indicates missing values.
    Figure Legend Snippet: Heatmap visualization of quantitative values of the 182 proteins enriched at the protein level . Left: heatmap of the 182 proteins identified and quantified over all six HNE exposure groups; Right: Zoomed-in region of the heatmap focusing on the most abundantly detected and identified putative HNE protein adducts. After affinity capture, samples were trypsin-digested and analyzed using LC-MS. Protein quantification was based on the peak intensity of the 3 most intense peptides. A total of 182 proteins were quantified. From top to bottom the 182 identified proteins are listed and each row represents a protein and its corresponding abundance. From left to right, HNE concentrations that were used for the in vitro exposure experiments of the mitochondrial protein samples. The color in each cell represents protein abundance obtained from the “Hi3” peptide intensity approach: red is more abundant and dark green is less abundant. Black indicates missing values.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, In Vitro

    7) Product Images from "Function of the CysD domain of the gel-forming MUC2 mucin"

    Article Title: Function of the CysD domain of the gel-forming MUC2 mucin

    Journal: Biochemical Journal

    doi: 10.1042/BJ20102066

    LC-ESI MS/MS analysis of the CysD peptide with a C-mannosylation motif A CysD-IgG-containing band from a non-reduced SDS gel was excised, in-gel digested with Asp-N and analysed by LC-ESI MS/MS. CID-fragmentation spectra of the CysD peptide DLSSPCVPLCNWTGWL in ( A ) with an intact disulfide bridge and in ( B ) after reduction with TCEP-HCl. Both fragmentation spectra showed the absence of C-mannosylation on the first tryptophan residue of the WXXW peptide motif.
    Figure Legend Snippet: LC-ESI MS/MS analysis of the CysD peptide with a C-mannosylation motif A CysD-IgG-containing band from a non-reduced SDS gel was excised, in-gel digested with Asp-N and analysed by LC-ESI MS/MS. CID-fragmentation spectra of the CysD peptide DLSSPCVPLCNWTGWL in ( A ) with an intact disulfide bridge and in ( B ) after reduction with TCEP-HCl. Both fragmentation spectra showed the absence of C-mannosylation on the first tryptophan residue of the WXXW peptide motif.

    Techniques Used: Mass Spectrometry, SDS-Gel

    8) Product Images from "A genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis †Electronic supplementary information (ESI) available: Fig. S1–S21; Tables S1–S5, full experimental details and procedures. See DOI: 10.1039/c5sc03059eClick here for additional data file."

    Article Title: A genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis genomics-led approach to deciphering the mechanism of thiotetronate antibiotic biosynthesis †Electronic supplementary information (ESI) available: Fig. S1–S21; Tables S1–S5, full experimental details and procedures. See DOI: 10.1039/c5sc03059eClick here for additional data file.

    Journal: Chemical Science

    doi: 10.1039/c5sc03059e

    HPLC-UV, LC-ESI-HRMS and MS–MS analysis of P450 deletion mutants from both Tü 3010 and thiolactomycin (TLM) biosynthetic pathways. (A) HPLC trace profiles (UV 238 nm ) of extracts from S. thiolactonus wild-type strain NRRL 15439 and mutants ΔstuD1 and ΔstuD2, the Lentzea sp. wild-type strain ATCC31319 and ΔtlmD1 mutant. Separation was achieved as described in the materials and methods section. Production of Tü 3010 (retention time 8.39 min) was abolished in both P450 ( stuD1 and stuD2 ) mutants, although the ΔstuD2 mutant produced a new UV-absorbing peak (retention time 10.93 min). Thiolactomycin (retention time 27.14 min) production was lost, with no obvious new UV-absorbing peak, upon disruption of tlmD1 . (B) Further LC-ESI-HRMS analysis of the ΔstuD2 intermediate by selective ion monitoring confirmed it as thiotetromycin ( 2 ) (Fig. S20 † ). Asterisk denotes not detected.
    Figure Legend Snippet: HPLC-UV, LC-ESI-HRMS and MS–MS analysis of P450 deletion mutants from both Tü 3010 and thiolactomycin (TLM) biosynthetic pathways. (A) HPLC trace profiles (UV 238 nm ) of extracts from S. thiolactonus wild-type strain NRRL 15439 and mutants ΔstuD1 and ΔstuD2, the Lentzea sp. wild-type strain ATCC31319 and ΔtlmD1 mutant. Separation was achieved as described in the materials and methods section. Production of Tü 3010 (retention time 8.39 min) was abolished in both P450 ( stuD1 and stuD2 ) mutants, although the ΔstuD2 mutant produced a new UV-absorbing peak (retention time 10.93 min). Thiolactomycin (retention time 27.14 min) production was lost, with no obvious new UV-absorbing peak, upon disruption of tlmD1 . (B) Further LC-ESI-HRMS analysis of the ΔstuD2 intermediate by selective ion monitoring confirmed it as thiotetromycin ( 2 ) (Fig. S20 † ). Asterisk denotes not detected.

    Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry, Mutagenesis, Produced

    9) Product Images from "Protein composition analysis of polyhedra matrix of Bombyx mori nucleopolyhedrovirus (BmNPV) showed powerful capacity of polyhedra to encapsulate foreign proteins"

    Article Title: Protein composition analysis of polyhedra matrix of Bombyx mori nucleopolyhedrovirus (BmNPV) showed powerful capacity of polyhedra to encapsulate foreign proteins

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08987-8

    BmNPV polyhedra matrix proteins were separated on 12% ( A ) and 15% ( B ) SDS-PAGE gels. Purified polyhedra were treated with an alkaline solution. Undissolved polyhedra were removed by low-speed centrifugation at 500 × g. The resulting supernatant was collected and then centrifuged by continuous sucrose gradients to remove the ODVs. Protein sample over the upper gradient was separated by SDS-PAGE. Proteins on the gel were excised into five contiguous sections (M1 to M5) and subjected to in-gel digestion and LC-ESI-MS/MS analysis. Two M5 sections shown in ( A ) and ( B ) were combined for the determination. Lane M, protein marker.
    Figure Legend Snippet: BmNPV polyhedra matrix proteins were separated on 12% ( A ) and 15% ( B ) SDS-PAGE gels. Purified polyhedra were treated with an alkaline solution. Undissolved polyhedra were removed by low-speed centrifugation at 500 × g. The resulting supernatant was collected and then centrifuged by continuous sucrose gradients to remove the ODVs. Protein sample over the upper gradient was separated by SDS-PAGE. Proteins on the gel were excised into five contiguous sections (M1 to M5) and subjected to in-gel digestion and LC-ESI-MS/MS analysis. Two M5 sections shown in ( A ) and ( B ) were combined for the determination. Lane M, protein marker.

    Techniques Used: SDS Page, Purification, Centrifugation, Mass Spectrometry, Marker

    10) Product Images from "A chloroquine-induced macrophage-preconditioning strategy for improved nanodelivery"

    Article Title: A chloroquine-induced macrophage-preconditioning strategy for improved nanodelivery

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14221-2

    Chloroquine-induced changes in Kupffer cells. ( a ) Microscopy images of chloroquine-induced vacuole formation in live cells. Cells were pretreated with 100 μM of chloroquine. Lysotracker, green. Scale bar, 50 μm (upper), 10 μm (lower). ( b ) Western blot analysis of phosphatidylinositol-binding clathrin assembly protein (PICALM), α-adaptin, and clathrin heavy chain expression in cells. β-actin was used as a loading control. ( c ) Densitometric analysis of western blot results. Results represent the ratio between the protein of interest and β-actin (mean ± s.d. of three samples), and are normalized to control cells. Statistics by Student’s t -test. *** P
    Figure Legend Snippet: Chloroquine-induced changes in Kupffer cells. ( a ) Microscopy images of chloroquine-induced vacuole formation in live cells. Cells were pretreated with 100 μM of chloroquine. Lysotracker, green. Scale bar, 50 μm (upper), 10 μm (lower). ( b ) Western blot analysis of phosphatidylinositol-binding clathrin assembly protein (PICALM), α-adaptin, and clathrin heavy chain expression in cells. β-actin was used as a loading control. ( c ) Densitometric analysis of western blot results. Results represent the ratio between the protein of interest and β-actin (mean ± s.d. of three samples), and are normalized to control cells. Statistics by Student’s t -test. *** P

    Techniques Used: Microscopy, Western Blot, Binding Assay, Expressing

    Effect of chloroquine on nanoparticle uptake in macrophages. ( a ) Viability of Raw 264.7, J774A.1, and Kupffer cells in response to chloroquine and nanoparticles. The left side of the dashed line indicates drug concentrations used in the nanoparticle uptake study. ( b ) Suppression of nanoparticle uptake upon exposure to chloroquine in Raw 264.7, J774A.1, and Kupffer cells. Values are normalized to those of control cells. Data is presented as mean ± s.d. of triplicates. Statistics by Student’s t -test. * P
    Figure Legend Snippet: Effect of chloroquine on nanoparticle uptake in macrophages. ( a ) Viability of Raw 264.7, J774A.1, and Kupffer cells in response to chloroquine and nanoparticles. The left side of the dashed line indicates drug concentrations used in the nanoparticle uptake study. ( b ) Suppression of nanoparticle uptake upon exposure to chloroquine in Raw 264.7, J774A.1, and Kupffer cells. Values are normalized to those of control cells. Data is presented as mean ± s.d. of triplicates. Statistics by Student’s t -test. * P

    Techniques Used:

    Effect of chloroquine on nanoparticle uptake in cancer cells. ( a ) Comparison of liposome (non-pegylated) uptake in macrophages (Raw 264.7, J774A.1, and Kupffer cells) and cancer cells (MDA-MB-231 breast cancer cells, MIA PaCa-2 pancreatic cancer cells, H358 lung cancer cells). ( b ) Viability of cancer cells in response to chloroquine and liposomes (6 h). ( c ) Effect of chloroquine on liposome uptake in MDA-MB-231 cells (6 h). Values are normalized to those of control cells. Data is presented as mean ± s.d. of triplicates. Statistics by Student’s t -test. ** P
    Figure Legend Snippet: Effect of chloroquine on nanoparticle uptake in cancer cells. ( a ) Comparison of liposome (non-pegylated) uptake in macrophages (Raw 264.7, J774A.1, and Kupffer cells) and cancer cells (MDA-MB-231 breast cancer cells, MIA PaCa-2 pancreatic cancer cells, H358 lung cancer cells). ( b ) Viability of cancer cells in response to chloroquine and liposomes (6 h). ( c ) Effect of chloroquine on liposome uptake in MDA-MB-231 cells (6 h). Values are normalized to those of control cells. Data is presented as mean ± s.d. of triplicates. Statistics by Student’s t -test. ** P

    Techniques Used: Multiple Displacement Amplification

    Effect of chloroquine on the biodistribution of liposomes. ( a ) Immunofluorescence staining of macrophages in the liver. Athymic nude mice were treated with clodronate liposomes (clodrolip; 50 mg/kg clodronate i.v.) or chloroquine (60 mg/kg/day i.p. for 7 days). DAPI, blue; macrophages (F4/80), green. Scale bar, 50 μm. ( b ) Accumulation of intravenously injected fluorescent liposomes in the plasma, liver, and spleen. The blood was collected and the liver and spleen were harvested 15 min, 3 h, 6 h, or 24 h post-injection of liposomes. ( c,d,e ) Effect of Kupffer cell depletion (clodrolip; 50 mg/kg clodronate) and chloroquine pretreatment (60 mg/kg/day for 7 days) on the biodistribution of fluorescent liposomes. The blood was collected and the organs were harvested 6 h post-injection of liposomes. (c) Detected signal in the plasma, liver, and spleen. ( d ) Plasma/liver and plasma/spleen accumulation ratio of liposomes (g tissue). ( e ) Accumulation of fluorescent liposomes in MDA-MB-231 orthotopic breast cancer tumors. Data is presented as mean ± s.d. ( n = 5). Statistics by Student’s t -test. * P
    Figure Legend Snippet: Effect of chloroquine on the biodistribution of liposomes. ( a ) Immunofluorescence staining of macrophages in the liver. Athymic nude mice were treated with clodronate liposomes (clodrolip; 50 mg/kg clodronate i.v.) or chloroquine (60 mg/kg/day i.p. for 7 days). DAPI, blue; macrophages (F4/80), green. Scale bar, 50 μm. ( b ) Accumulation of intravenously injected fluorescent liposomes in the plasma, liver, and spleen. The blood was collected and the liver and spleen were harvested 15 min, 3 h, 6 h, or 24 h post-injection of liposomes. ( c,d,e ) Effect of Kupffer cell depletion (clodrolip; 50 mg/kg clodronate) and chloroquine pretreatment (60 mg/kg/day for 7 days) on the biodistribution of fluorescent liposomes. The blood was collected and the organs were harvested 6 h post-injection of liposomes. (c) Detected signal in the plasma, liver, and spleen. ( d ) Plasma/liver and plasma/spleen accumulation ratio of liposomes (g tissue). ( e ) Accumulation of fluorescent liposomes in MDA-MB-231 orthotopic breast cancer tumors. Data is presented as mean ± s.d. ( n = 5). Statistics by Student’s t -test. * P

    Techniques Used: Immunofluorescence, Staining, Mouse Assay, Injection, Multiple Displacement Amplification

    Nanoparticle characterization and uptake in macrophages. ( a ) Characterization of nanoparticles. ( b ) Schematic illustration of the various pathways of nanoparticle uptake in macrophages. Inhibitors of specific pathways are shown. Cytochalasin D was used as a broad-spectrum inhibitor of actin-dependent uptake. ( c ) Nanoparticle uptake in macrophages. Suppression of nanoparticle uptake upon exposure to cytochalasin D (bar graph ‘+’) in Raw 264.7, J774A.1, and Kupffer cells. Values are normalized to those of control cells. Data is presented as mean ± s.d. of triplicates. Statistics by Student’s t -test. * P
    Figure Legend Snippet: Nanoparticle characterization and uptake in macrophages. ( a ) Characterization of nanoparticles. ( b ) Schematic illustration of the various pathways of nanoparticle uptake in macrophages. Inhibitors of specific pathways are shown. Cytochalasin D was used as a broad-spectrum inhibitor of actin-dependent uptake. ( c ) Nanoparticle uptake in macrophages. Suppression of nanoparticle uptake upon exposure to cytochalasin D (bar graph ‘+’) in Raw 264.7, J774A.1, and Kupffer cells. Values are normalized to those of control cells. Data is presented as mean ± s.d. of triplicates. Statistics by Student’s t -test. * P

    Techniques Used:

    11) Product Images from "Crosstalk among the proteome, lysine phosphorylation, and acetylation in romidepsin-treated colon cancer cells"

    Article Title: Crosstalk among the proteome, lysine phosphorylation, and acetylation in romidepsin-treated colon cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10840

    Changes in the proteome profile following FK228 treatment between HCT-8 and HCT-116 cells Quantitative lysine-acetylome and global-phosphorylation analyses were performed in HCT-8 and HCT-116 cells using SILAC and affinity enrichment, followed by high-resolution LC-MS/MS analysis. Total and differentially modified proteins and regulation patterns were identified. ( A ) Differentially regulated protein groups between the two cell lines. ( B ) Differential regulation patterns between the two cell lines.
    Figure Legend Snippet: Changes in the proteome profile following FK228 treatment between HCT-8 and HCT-116 cells Quantitative lysine-acetylome and global-phosphorylation analyses were performed in HCT-8 and HCT-116 cells using SILAC and affinity enrichment, followed by high-resolution LC-MS/MS analysis. Total and differentially modified proteins and regulation patterns were identified. ( A ) Differentially regulated protein groups between the two cell lines. ( B ) Differential regulation patterns between the two cell lines.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification

    Phosphorylation profile changes following FK228 treatment of HCT-8 and HCT-116 cells SILAC, affinity enrichment, and high-resolution LC-MS/MS analysis was used for quantitative phosphoproteomics analysis of HCT-8 and HCT-116 cells following FK228 treatment. Total and differentially phosphorylated sites and phosphorylated proteins were assessed. ( A ) Sites exhibiting differential phosphorylation patterns in the two cell lines. ( B ) Differentially phosphorylated proteins between the two cell lines.
    Figure Legend Snippet: Phosphorylation profile changes following FK228 treatment of HCT-8 and HCT-116 cells SILAC, affinity enrichment, and high-resolution LC-MS/MS analysis was used for quantitative phosphoproteomics analysis of HCT-8 and HCT-116 cells following FK228 treatment. Total and differentially phosphorylated sites and phosphorylated proteins were assessed. ( A ) Sites exhibiting differential phosphorylation patterns in the two cell lines. ( B ) Differentially phosphorylated proteins between the two cell lines.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    12) Product Images from "An Alternative Strategy for Pan-acetyl-lysine Antibody Generation"

    Article Title: An Alternative Strategy for Pan-acetyl-lysine Antibody Generation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0162528

    Strategy for pan-acetyl-lysine antibody generation and application in acetylome studies. To generate pan-acetyl-lysine (acK) antibody, a random acK peptide library conjugated to ovalbumin was used as an antigen to immunize rabbits. Raised antibodies were tested for specificity with ELISA and dot blot, and those that passed quality control were used to pull down acK peptides from HEK293 cell lysate for analysis by LC-MS/MS. A commercial pan-acK antibody was used for comparison.
    Figure Legend Snippet: Strategy for pan-acetyl-lysine antibody generation and application in acetylome studies. To generate pan-acetyl-lysine (acK) antibody, a random acK peptide library conjugated to ovalbumin was used as an antigen to immunize rabbits. Raised antibodies were tested for specificity with ELISA and dot blot, and those that passed quality control were used to pull down acK peptides from HEK293 cell lysate for analysis by LC-MS/MS. A commercial pan-acK antibody was used for comparison.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Dot Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Identification of acetylated peptides by LC-MS/MS and their consensus sequence motif. Overlap of identified acetylated proteins (A) and peptides (B) from HEK293 cells using commercial pan-acK antibody or SICS pan-acK antibodies. (C and D) Peptide sequence motif and heat map of amino acids flanking the acetyl-lysine ( P
    Figure Legend Snippet: Identification of acetylated peptides by LC-MS/MS and their consensus sequence motif. Overlap of identified acetylated proteins (A) and peptides (B) from HEK293 cells using commercial pan-acK antibody or SICS pan-acK antibodies. (C and D) Peptide sequence motif and heat map of amino acids flanking the acetyl-lysine ( P

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing

    13) Product Images from "A Double-Barrel Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) System to Quantify 96 Interactomes per Day *"

    Article Title: A Double-Barrel Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) System to Quantify 96 Interactomes per Day *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.O115.049460

    Workflow of the high-throughput LC-MS/MS protein interaction analysis pipeline. Both culturing of yeast cells and affinity purification are performed in 96-well plate format, thus parallelizing sample preparation and minimizing handling errors. LC-MS/MS analysis of 96 pull-down samples in 1 day is achieved through a double-barrel chromatography setup and the increased sequencing speed of the Q Exactive HF mass spectrometer.
    Figure Legend Snippet: Workflow of the high-throughput LC-MS/MS protein interaction analysis pipeline. Both culturing of yeast cells and affinity purification are performed in 96-well plate format, thus parallelizing sample preparation and minimizing handling errors. LC-MS/MS analysis of 96 pull-down samples in 1 day is achieved through a double-barrel chromatography setup and the increased sequencing speed of the Q Exactive HF mass spectrometer.

    Techniques Used: High Throughput Screening Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Affinity Purification, Sample Prep, Chromatography, Sequencing

    Double-barrel chromatography with 14 min gradients on three pull-downs. ( A ) Base-peak chromatogram of a biological triplicate RSC8 pull-down run on the double-barrel LC-MS/MS setup. Chromatography in all cases is very reproducible. ( B ) Comparison of RSC8, SPT7 and SWI3 pull-downs; all measured in triplicates. The matrix of 36 correlation plots reveals high correlations between MaxLFQ intensities within triplicates. ( C ) Zoom into SPT7_02 versus the SWI3_01 correlation plot. While most proteins were detected with very similar MaxLFQ intensities, the two outlier populations marked in orange (SPT7) and blue (SWI3) represent the different complex members of the distinct protein complexes.
    Figure Legend Snippet: Double-barrel chromatography with 14 min gradients on three pull-downs. ( A ) Base-peak chromatogram of a biological triplicate RSC8 pull-down run on the double-barrel LC-MS/MS setup. Chromatography in all cases is very reproducible. ( B ) Comparison of RSC8, SPT7 and SWI3 pull-downs; all measured in triplicates. The matrix of 36 correlation plots reveals high correlations between MaxLFQ intensities within triplicates. ( C ) Zoom into SPT7_02 versus the SWI3_01 correlation plot. While most proteins were detected with very similar MaxLFQ intensities, the two outlier populations marked in orange (SPT7) and blue (SWI3) represent the different complex members of the distinct protein complexes.

    Techniques Used: Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    14) Product Images from "Potent Mechanism-Based Inactivation Of Cytochrome P450 2B4 By 9-Ethynylphenanthrene: Implications For Allosteric Modulation Of Cytochrome P450 Catalysis"

    Article Title: Potent Mechanism-Based Inactivation Of Cytochrome P450 2B4 By 9-Ethynylphenanthrene: Implications For Allosteric Modulation Of Cytochrome P450 Catalysis

    Journal: Biochemistry

    doi: 10.1021/bi301567z

    Analysis of the molecular mass of the 9EP-inactivated CYP2B4 using ESI-LC/MS. CYP2B4 (1 μM) was inactivated by 10 μM 9EP in 50 mM KPi buffer (pH 7.4) in the presence of 0.5 μM CPR, 3 μM cyt b5 and 1 mM NADPH at 30 °C
    Figure Legend Snippet: Analysis of the molecular mass of the 9EP-inactivated CYP2B4 using ESI-LC/MS. CYP2B4 (1 μM) was inactivated by 10 μM 9EP in 50 mM KPi buffer (pH 7.4) in the presence of 0.5 μM CPR, 3 μM cyt b5 and 1 mM NADPH at 30 °C

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    15) Product Images from "Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling"

    Article Title: Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling

    Journal: Biochemical Journal

    doi: 10.1042/BCJ20160740

    Mass spectrometry identifies phosphorylation sites in PTH1R. ( A ) HEK293T cells were transiently transfected with HA-tagged PTH1R and labeled with [ 32 P]orthophosphate followed by immunoprecipitation. HEK293 cells stably transfected with PTH1R–HA were used to immunoprecipitate PTH1R, which was then digested with trypsin and analyzed by mass spectrometry. For 32 P labeling and mass spectrometry studies, cells were stimulated with 500 nM PTH(1–34) for 8 min. Left panel: autoradiograph and Western blot (anti-HA antibody) loading control. Right panel: levels of 32 P were quantified by densitometry and are presented as fold increases in phosphorylation relative to non-stimulated controls. Data are representative of three independent experiments ± SEM. ( B ) Schematic representation of PTH1R. Locations of phosphorylation sites identified by MS/MS are indicated in red, and red boxes indicate the positions of phosphorylation site clusters 1 and 2. ( C ) Representative MS/MS spectra and associated fragmentation tables for two PTH1R phosphopeptides are shown. ( D ) Primary amino acid sequence of PTH1R indicating in red the amino acids identified as being phosphorylated and underlined in black are the regions of PTH1R covered by the analysis.
    Figure Legend Snippet: Mass spectrometry identifies phosphorylation sites in PTH1R. ( A ) HEK293T cells were transiently transfected with HA-tagged PTH1R and labeled with [ 32 P]orthophosphate followed by immunoprecipitation. HEK293 cells stably transfected with PTH1R–HA were used to immunoprecipitate PTH1R, which was then digested with trypsin and analyzed by mass spectrometry. For 32 P labeling and mass spectrometry studies, cells were stimulated with 500 nM PTH(1–34) for 8 min. Left panel: autoradiograph and Western blot (anti-HA antibody) loading control. Right panel: levels of 32 P were quantified by densitometry and are presented as fold increases in phosphorylation relative to non-stimulated controls. Data are representative of three independent experiments ± SEM. ( B ) Schematic representation of PTH1R. Locations of phosphorylation sites identified by MS/MS are indicated in red, and red boxes indicate the positions of phosphorylation site clusters 1 and 2. ( C ) Representative MS/MS spectra and associated fragmentation tables for two PTH1R phosphopeptides are shown. ( D ) Primary amino acid sequence of PTH1R indicating in red the amino acids identified as being phosphorylated and underlined in black are the regions of PTH1R covered by the analysis.

    Techniques Used: Mass Spectrometry, Transfection, Labeling, Immunoprecipitation, Stable Transfection, Autoradiography, Western Blot, Sequencing

    16) Product Images from "Protein composition analysis of polyhedra matrix of Bombyx mori nucleopolyhedrovirus (BmNPV) showed powerful capacity of polyhedra to encapsulate foreign proteins"

    Article Title: Protein composition analysis of polyhedra matrix of Bombyx mori nucleopolyhedrovirus (BmNPV) showed powerful capacity of polyhedra to encapsulate foreign proteins

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08987-8

    BmNPV polyhedra matrix proteins were separated on 12% ( A ) and 15% ( B ) SDS-PAGE gels. Purified polyhedra were treated with an alkaline solution. Undissolved polyhedra were removed by low-speed centrifugation at 500 × g. The resulting supernatant was collected and then centrifuged by continuous sucrose gradients to remove the ODVs. Protein sample over the upper gradient was separated by SDS-PAGE. Proteins on the gel were excised into five contiguous sections (M1 to M5) and subjected to in-gel digestion and LC-ESI-MS/MS analysis. Two M5 sections shown in ( A ) and ( B ) were combined for the determination. Lane M, protein marker.
    Figure Legend Snippet: BmNPV polyhedra matrix proteins were separated on 12% ( A ) and 15% ( B ) SDS-PAGE gels. Purified polyhedra were treated with an alkaline solution. Undissolved polyhedra were removed by low-speed centrifugation at 500 × g. The resulting supernatant was collected and then centrifuged by continuous sucrose gradients to remove the ODVs. Protein sample over the upper gradient was separated by SDS-PAGE. Proteins on the gel were excised into five contiguous sections (M1 to M5) and subjected to in-gel digestion and LC-ESI-MS/MS analysis. Two M5 sections shown in ( A ) and ( B ) were combined for the determination. Lane M, protein marker.

    Techniques Used: SDS Page, Purification, Centrifugation, Mass Spectrometry, Marker

    17) Product Images from "Biological Activities of Aerial Parts Extracts of Euphorbia characias"

    Article Title: Biological Activities of Aerial Parts Extracts of Euphorbia characias

    Journal: BioMed Research International

    doi: 10.1155/2016/1538703

    Identification of polyphenolic compounds in E. characias leaves using LC-ESI-Orbitrap-MS/MS in negative ion mode. Chromatographic conditions are described in the text. A list of compounds is reported in Table 5 .
    Figure Legend Snippet: Identification of polyphenolic compounds in E. characias leaves using LC-ESI-Orbitrap-MS/MS in negative ion mode. Chromatographic conditions are described in the text. A list of compounds is reported in Table 5 .

    Techniques Used: Mass Spectrometry

    18) Product Images from "Mass Spectrometry of Structurally Modified DNA"

    Article Title: Mass Spectrometry of Structurally Modified DNA

    Journal: Chemical reviews

    doi: 10.1021/cr300391r

    Chromatograms obtained upon LC-NSI-HRMS/MS analysis of human leukocyte DNA (129 μg, 12.9 μg on column) containing 59.4 fmol N7-ethyl-Gua /μmol Gua. The relatively higher amount of analyte in this sample allowed for the confirmation
    Figure Legend Snippet: Chromatograms obtained upon LC-NSI-HRMS/MS analysis of human leukocyte DNA (129 μg, 12.9 μg on column) containing 59.4 fmol N7-ethyl-Gua /μmol Gua. The relatively higher amount of analyte in this sample allowed for the confirmation

    Techniques Used: Mass Spectrometry

    19) Product Images from "Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1"

    Article Title: Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1

    Journal: Journal of the American Society for Mass Spectrometry

    doi: 10.1007/s13361-016-1379-z

    The Q-Exactive plus Orbitrap EMR mass spectrometer produces highly resolved spectra which distinguish between the three DJ-1 variants.
    Figure Legend Snippet: The Q-Exactive plus Orbitrap EMR mass spectrometer produces highly resolved spectra which distinguish between the three DJ-1 variants.

    Techniques Used: Mass Spectrometry

    20) Product Images from "Production of the Plant Hormone Auxin by Salmonella and Its Role in the Interactions with Plants and Animals"

    Article Title: Production of the Plant Hormone Auxin by Salmonella and Its Role in the Interactions with Plants and Animals

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02668

    Production of IAA in ipdC -dependent manner by Salmonella enterica sv. Typhimuirum 14028. (A) Detection of IAA by the Salkowski reagent. Results are from replicates from three independent experiments, averages are shown. Error bars are standard deviations. (B) HPLC detection of IAA and tryptophol in spent cultures of the wild type and the ipdC mutant. Hydrophobic fractions of Salmonella culture filtrates were subjected to reverse phase (C 18 ) liquid chromatography, eluted isocratically with acidified water/methanol and eluting substances were detected with a UV/VIS detector set to 230 and 280 nm. (C) Low Resolution Electrospray Ionization LC-MS of the hydrophobic fraction of Salmonella culture filtrates. LTQ LC-MS was carried out using a Grace Vydac 218TP C18 and acetonitrile/water gradient. (D) Expression of the ipdC RIVET reporter in the wild type and Δ ipdC backgrounds was measured in Minimal A medium with and without synthetic IAA or tryptophan in cultures incubated at 22°C for 72 h. Samples were streaked to xylose lysine deoxycholate (XLD) agar with kanamycin at 24 and 72 h. Plates were incubated at 37°C overnight and colonies were patched to LB agar with tetracycline to quantify resolution. Experiments were repeated five times (without technical replications), averages from the five experiments are shown. Error bars are standard deviations.
    Figure Legend Snippet: Production of IAA in ipdC -dependent manner by Salmonella enterica sv. Typhimuirum 14028. (A) Detection of IAA by the Salkowski reagent. Results are from replicates from three independent experiments, averages are shown. Error bars are standard deviations. (B) HPLC detection of IAA and tryptophol in spent cultures of the wild type and the ipdC mutant. Hydrophobic fractions of Salmonella culture filtrates were subjected to reverse phase (C 18 ) liquid chromatography, eluted isocratically with acidified water/methanol and eluting substances were detected with a UV/VIS detector set to 230 and 280 nm. (C) Low Resolution Electrospray Ionization LC-MS of the hydrophobic fraction of Salmonella culture filtrates. LTQ LC-MS was carried out using a Grace Vydac 218TP C18 and acetonitrile/water gradient. (D) Expression of the ipdC RIVET reporter in the wild type and Δ ipdC backgrounds was measured in Minimal A medium with and without synthetic IAA or tryptophan in cultures incubated at 22°C for 72 h. Samples were streaked to xylose lysine deoxycholate (XLD) agar with kanamycin at 24 and 72 h. Plates were incubated at 37°C overnight and colonies were patched to LB agar with tetracycline to quantify resolution. Experiments were repeated five times (without technical replications), averages from the five experiments are shown. Error bars are standard deviations.

    Techniques Used: High Performance Liquid Chromatography, Mutagenesis, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Expressing, Incubation

    21) Product Images from "Reaction rate of pyruvate and hydrogen peroxide: assessing antioxidant capacity of pyruvate under biological conditions"

    Article Title: Reaction rate of pyruvate and hydrogen peroxide: assessing antioxidant capacity of pyruvate under biological conditions

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-55951-9

    LC-MS measurement of pyruvate and acetate concentrations. Reactions were carried out with 300 µM each of pyruvate and H 2 O 2 in DPBS at 37 °C. At the indicated time, aliquots of the reaction mixture were removed for LC-MS analysis. (A , B) LC-MS chromatograms showing the detection of pyruvate and acetate, respectively, in the samples at the indicated times. (C , D) Concentrations of pyruvate and acetate, respectively, were calculated according to standard curves, graphed vs. time, and fit to second-degree polynomial equations. (E) Data are graphed as the sum of pyruvate and acetate concentrations vs. time, showing an unchanged total concentration over time. (F) Data are graphed as the inverse concentration of pyruvate vs. time and fit to a linear equation. The slope of the line indicates the k of the reaction. Data were obtained from 3 separate experiments and presented as mean ± SD.
    Figure Legend Snippet: LC-MS measurement of pyruvate and acetate concentrations. Reactions were carried out with 300 µM each of pyruvate and H 2 O 2 in DPBS at 37 °C. At the indicated time, aliquots of the reaction mixture were removed for LC-MS analysis. (A , B) LC-MS chromatograms showing the detection of pyruvate and acetate, respectively, in the samples at the indicated times. (C , D) Concentrations of pyruvate and acetate, respectively, were calculated according to standard curves, graphed vs. time, and fit to second-degree polynomial equations. (E) Data are graphed as the sum of pyruvate and acetate concentrations vs. time, showing an unchanged total concentration over time. (F) Data are graphed as the inverse concentration of pyruvate vs. time and fit to a linear equation. The slope of the line indicates the k of the reaction. Data were obtained from 3 separate experiments and presented as mean ± SD.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Concentration Assay

    22) Product Images from "LipidBlast - in-silico tandem mass spectrometry database for lipid identification"

    Article Title: LipidBlast - in-silico tandem mass spectrometry database for lipid identification

    Journal: Nature methods

    doi: 10.1038/nmeth.2551

    Creation, validation and application of in-silico generated tandem mass spectra in LipidBlast (a) New lipid compound structures were created using combinatorial chemistry approaches. A scaffold of the lipid core structure and linker are connected to fatty acyls with different chain lengths and different degrees of unsaturation. (b) The reference tandem spectra (upper panel) are used to simulate the mass spectral fragmentations and ion abundances of the in-silico spectra (lower panel). The compound shown here is phosphatidylcholine PC(16:0/16:1) at precursor m/z =732.55 [M+H] + . (c) T andem mass spectra are obtained from LC-MS/MS or direct-infusion experiments. The MS/MS spectra are submitted to LipidBlast MS/MS search. An m/z precursor ion filter serves as first powerful filter and a subsequent product ion match creates a library hit score that is related to the level of confidence for the compound annotation.
    Figure Legend Snippet: Creation, validation and application of in-silico generated tandem mass spectra in LipidBlast (a) New lipid compound structures were created using combinatorial chemistry approaches. A scaffold of the lipid core structure and linker are connected to fatty acyls with different chain lengths and different degrees of unsaturation. (b) The reference tandem spectra (upper panel) are used to simulate the mass spectral fragmentations and ion abundances of the in-silico spectra (lower panel). The compound shown here is phosphatidylcholine PC(16:0/16:1) at precursor m/z =732.55 [M+H] + . (c) T andem mass spectra are obtained from LC-MS/MS or direct-infusion experiments. The MS/MS spectra are submitted to LipidBlast MS/MS search. An m/z precursor ion filter serves as first powerful filter and a subsequent product ion match creates a library hit score that is related to the level of confidence for the compound annotation.

    Techniques Used: In Silico, Generated, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    23) Product Images from "Proteomic Consequences of a Single Gene Mutation in a Colorectal Cancer Model"

    Article Title: Proteomic Consequences of a Single Gene Mutation in a Colorectal Cancer Model

    Journal: Journal of Proteome Research

    doi: 10.1021/pr2009109

    Proteomic changes associated with APC restoration. (A) Schematic figure represents a subset of enriched classes from 155 differentially expressed proteins (
    Figure Legend Snippet: Proteomic changes associated with APC restoration. (A) Schematic figure represents a subset of enriched classes from 155 differentially expressed proteins (

    Techniques Used:

    Validation of proteomic differences by LC–MRM-MS. Shotgun proteomics data are plotted as spectral counts for triplicate analyses (red bars), whereas MRM data are plotted as summed signal intensity for measured transitions normalized to summed intensity for transitions measured for a reference peptide (blue bars). ( n = 3). A list of peptides and corresponding precursor and product m / z values is provided in Supplementary Table S2.
    Figure Legend Snippet: Validation of proteomic differences by LC–MRM-MS. Shotgun proteomics data are plotted as spectral counts for triplicate analyses (red bars), whereas MRM data are plotted as summed signal intensity for measured transitions normalized to summed intensity for transitions measured for a reference peptide (blue bars). ( n = 3). A list of peptides and corresponding precursor and product m / z values is provided in Supplementary Table S2.

    Techniques Used: Mass Spectrometry

    Biological variation of LC−MS/MS proteomics. (A) Venn diagrams show protein expression overlap for shotgun proteomic inventories in SW480APC and SW480Null (top). Overlap in three biological replicates is shown for SW480APC (bottom left) and SW480Null (bottom right). Spearman ranked correlations for replicate to replicate comparisons shown in parentheses (B) Stacked plots show percentage of proteins identified in one, two or three biological replicates in SW80APC and SW480Null.
    Figure Legend Snippet: Biological variation of LC−MS/MS proteomics. (A) Venn diagrams show protein expression overlap for shotgun proteomic inventories in SW480APC and SW480Null (top). Overlap in three biological replicates is shown for SW480APC (bottom left) and SW480Null (bottom right). Spearman ranked correlations for replicate to replicate comparisons shown in parentheses (B) Stacked plots show percentage of proteins identified in one, two or three biological replicates in SW80APC and SW480Null.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing

    Proteomic and transcriptomic profile comparison of SW480APC and SW480Null. Heat map shows supervised clustering analysis of shotgun proteomics data and transcriptomic data for 111 differentially expressed proteins (adjusted quasi p -value
    Figure Legend Snippet: Proteomic and transcriptomic profile comparison of SW480APC and SW480Null. Heat map shows supervised clustering analysis of shotgun proteomics data and transcriptomic data for 111 differentially expressed proteins (adjusted quasi p -value

    Techniques Used:

    24) Product Images from "Aging‐associated changes in hippocampal glycogen metabolism in mice. Evidence for and against astrocyte‐to‐neuron lactate shuttle. Aging‐associated changes in hippocampal glycogen metabolism in mice. Evidence for and against astrocyte‐to‐neuron lactate shuttle"

    Article Title: Aging‐associated changes in hippocampal glycogen metabolism in mice. Evidence for and against astrocyte‐to‐neuron lactate shuttle. Aging‐associated changes in hippocampal glycogen metabolism in mice. Evidence for and against astrocyte‐to‐neuron lactate shuttle

    Journal: Glia

    doi: 10.1002/glia.23319

    Proteomic analysis of hippocampi from young and middle‐aged mice. (a) Proteomic workflow. Hippocampi were isolated from 6 animals per group. The tissue lysates were processed by MED‐FASP procedure with three steps of enzymatic digestion. Peptides were analyzed by LC‐MS/MS. The spectra were searched using MaxQuant software. Proteins were quantified by means of Total Protein Approach. (b) Protein to peptide conversion and peptide analysis. The numbers above the columns show the average number of peptides identified from each digest ( k , values are in thousands). (c) Number of proteins matching the peptides identified in the mass spectrometric analysis. 6,594 proteins were identified in at least 9 of 12 samples (valid values) and were used for the statistical analysis. (d) Distribution of protein copy numbers across the identified proteins [Color figure can be viewed at http://wileyonlinelibrary.com ]
    Figure Legend Snippet: Proteomic analysis of hippocampi from young and middle‐aged mice. (a) Proteomic workflow. Hippocampi were isolated from 6 animals per group. The tissue lysates were processed by MED‐FASP procedure with three steps of enzymatic digestion. Peptides were analyzed by LC‐MS/MS. The spectra were searched using MaxQuant software. Proteins were quantified by means of Total Protein Approach. (b) Protein to peptide conversion and peptide analysis. The numbers above the columns show the average number of peptides identified from each digest ( k , values are in thousands). (c) Number of proteins matching the peptides identified in the mass spectrometric analysis. 6,594 proteins were identified in at least 9 of 12 samples (valid values) and were used for the statistical analysis. (d) Distribution of protein copy numbers across the identified proteins [Color figure can be viewed at http://wileyonlinelibrary.com ]

    Techniques Used: Mouse Assay, Isolation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Software

    25) Product Images from "Comparative Proteomic Study of Fatty Acid-treated Myoblasts Reveals Role of Cox-2 in Palmitate-induced Insulin Resistance"

    Article Title: Comparative Proteomic Study of Fatty Acid-treated Myoblasts Reveals Role of Cox-2 in Palmitate-induced Insulin Resistance

    Journal: Scientific Reports

    doi: 10.1038/srep21454

    SILAC-based comparative proteomic analysis of fatty acid-treated myoblasts. ( A ) Flowcharts of forward SILAC and reverse SILAC labeling combined with LC-MS/MS for the comparative analysis of protein expression in C2C12 myoblasts upon PA and PA + OA treatment(R0/K0: normal isotopes, K4/R6: [ 2 H 4 ]lysine/[ 13 C 6 ] arginine, K8/R10: [ 13 C 6 , 15 N 2 ]lysine/[ 13 C 6 , 15 N 4 ] arginine). ( B) Quantitation overlap of the quantified proteins in the two forward and one reverse SILAC labeling experiments (F1 F2: forward experiment, R1: reverse experiment). ( C ) The distribution of expression ratios for the quantified proteins, including those quantified in only one set of SILAC labeling experiment, and the mean protein expression ratios were used for the data quantified in at least two sets). ( D ) (STRING database mapping the interaction of the differentially expressed proteins upon the treatment of PA and PA + OA. These proteins form several apparent interaction groups exerting diverse biological functions including ER stress, lipid metabolism, immune response, gene transcription and protein synthesis.
    Figure Legend Snippet: SILAC-based comparative proteomic analysis of fatty acid-treated myoblasts. ( A ) Flowcharts of forward SILAC and reverse SILAC labeling combined with LC-MS/MS for the comparative analysis of protein expression in C2C12 myoblasts upon PA and PA + OA treatment(R0/K0: normal isotopes, K4/R6: [ 2 H 4 ]lysine/[ 13 C 6 ] arginine, K8/R10: [ 13 C 6 , 15 N 2 ]lysine/[ 13 C 6 , 15 N 4 ] arginine). ( B) Quantitation overlap of the quantified proteins in the two forward and one reverse SILAC labeling experiments (F1 F2: forward experiment, R1: reverse experiment). ( C ) The distribution of expression ratios for the quantified proteins, including those quantified in only one set of SILAC labeling experiment, and the mean protein expression ratios were used for the data quantified in at least two sets). ( D ) (STRING database mapping the interaction of the differentially expressed proteins upon the treatment of PA and PA + OA. These proteins form several apparent interaction groups exerting diverse biological functions including ER stress, lipid metabolism, immune response, gene transcription and protein synthesis.

    Techniques Used: Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing, Quantitation Assay

    26) Product Images from "Convergence of Ubiquitylation and Phosphorylation Signaling in Rapamycin-treated Yeast Cells *"

    Article Title: Convergence of Ubiquitylation and Phosphorylation Signaling in Rapamycin-treated Yeast Cells *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.O113.035683

    Proteome, phosphoproteome, and ubiquitylome analysis of rapamycin-treated yeast. A , experimental outline. Exponentially growing yeast cells were metabolically labeled with lysine 0 (light), lysine 4 (medium), or lysine 8 (heavy). Rapamycin was added to 0.2 m m , and cells were harvested at the indicated time points. Equal amounts of proteins were mixed and digested under denaturing conditions using endoproteinase Lys-C. Phosphorylated peptides were enriched using TiO 2 -based chromatography, and di-Gly-modified (ubiquitylated) peptides were enriched using anti-di-Gly monoclonal antibody. All peptides were fractionated with micro-SCX prior to analysis using reversed phase liquid chromatography–tandem mass spectrometry (LC-MS/MS). B , overlap between biological replicates for proteome, phosphoproteome, and ubiquitylome. The Venn diagrams indicate the number ( n ) of sites or proteins identified in each experiment and the overlap between biological replicates.
    Figure Legend Snippet: Proteome, phosphoproteome, and ubiquitylome analysis of rapamycin-treated yeast. A , experimental outline. Exponentially growing yeast cells were metabolically labeled with lysine 0 (light), lysine 4 (medium), or lysine 8 (heavy). Rapamycin was added to 0.2 m m , and cells were harvested at the indicated time points. Equal amounts of proteins were mixed and digested under denaturing conditions using endoproteinase Lys-C. Phosphorylated peptides were enriched using TiO 2 -based chromatography, and di-Gly-modified (ubiquitylated) peptides were enriched using anti-di-Gly monoclonal antibody. All peptides were fractionated with micro-SCX prior to analysis using reversed phase liquid chromatography–tandem mass spectrometry (LC-MS/MS). B , overlap between biological replicates for proteome, phosphoproteome, and ubiquitylome. The Venn diagrams indicate the number ( n ) of sites or proteins identified in each experiment and the overlap between biological replicates.

    Techniques Used: Metabolic Labelling, Labeling, Chromatography, Modification, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    27) Product Images from "Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1"

    Article Title: Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1

    Journal: Journal of the American Society for Mass Spectrometry

    doi: 10.1007/s13361-016-1379-z

    The Q-Exactive plus Orbitrap EMR mass spectrometer produces highly resolved spectra which distinguish between the three DJ-1 variants.
    Figure Legend Snippet: The Q-Exactive plus Orbitrap EMR mass spectrometer produces highly resolved spectra which distinguish between the three DJ-1 variants.

    Techniques Used: Mass Spectrometry

    28) Product Images from "Effects of Caloric Restriction with Protein Supplementation on Plasma Protein Profiles in Middle-Aged Women with Metabolic Syndrome—A Preliminary Open Study"

    Article Title: Effects of Caloric Restriction with Protein Supplementation on Plasma Protein Profiles in Middle-Aged Women with Metabolic Syndrome—A Preliminary Open Study

    Journal: Journal of Clinical Medicine

    doi: 10.3390/jcm8020195

    Volcano plot of plasma selected proteomic ratio measurements between baseline and postintervention in the CR group. Fold change means the postintervention value divided by the baseline value, and the p -value was according to a Wilcoxon signed-rank test that compared the different protein expressions before and after the interventions within the groups. Thresholds are presented as dotted lines. The fold change cut-off points were 1.25 and 1/1.25, and the p -value cut-off point was 0.05. Both data were converted to a natural logarithm, as shown. CR: caloric restriction, A2M: alpha-2-macroglobulin, AMBP: alpha-1-microglobulin/bikunin precursor, APOA2: apolipoprotein A2, APOB: apolipoprotein B-100, APOC2: apolipoprotein C2, CO3: complement component 3, CO6: complement component 6, FIBA: fibrinogen alpha chain, FINC: fibronectin.
    Figure Legend Snippet: Volcano plot of plasma selected proteomic ratio measurements between baseline and postintervention in the CR group. Fold change means the postintervention value divided by the baseline value, and the p -value was according to a Wilcoxon signed-rank test that compared the different protein expressions before and after the interventions within the groups. Thresholds are presented as dotted lines. The fold change cut-off points were 1.25 and 1/1.25, and the p -value cut-off point was 0.05. Both data were converted to a natural logarithm, as shown. CR: caloric restriction, A2M: alpha-2-macroglobulin, AMBP: alpha-1-microglobulin/bikunin precursor, APOA2: apolipoprotein A2, APOB: apolipoprotein B-100, APOC2: apolipoprotein C2, CO3: complement component 3, CO6: complement component 6, FIBA: fibrinogen alpha chain, FINC: fibronectin.

    Techniques Used:

    Volcano plot of plasma selected proteomic ratio measurements between baseline and postintervention in the CRPS group. Fold change represents the postintervention value divided by the baseline value, and the p -value is associated with a Wilcoxon signed-rank test that compared the different protein expression levels before and after the interventions within the group. Thresholds are presented as dotted lines. The fold change cut-off points were 1.25 and 1/1.25, and the p -value cut-off point was 0.05. Both sets of data were converted to a natural logarithm as shown. CRPS: caloric restriction with protein supplementation, ALBU: albumin, FINC: fibronectin.
    Figure Legend Snippet: Volcano plot of plasma selected proteomic ratio measurements between baseline and postintervention in the CRPS group. Fold change represents the postintervention value divided by the baseline value, and the p -value is associated with a Wilcoxon signed-rank test that compared the different protein expression levels before and after the interventions within the group. Thresholds are presented as dotted lines. The fold change cut-off points were 1.25 and 1/1.25, and the p -value cut-off point was 0.05. Both sets of data were converted to a natural logarithm as shown. CRPS: caloric restriction with protein supplementation, ALBU: albumin, FINC: fibronectin.

    Techniques Used: Expressing

    29) Product Images from "Myeloperoxidase Targets Apolipoprotein A-I, the Major High Density Lipoprotein Protein, for Site-Specific Oxidation in Human Atherosclerotic Lesions *"

    Article Title: Myeloperoxidase Targets Apolipoprotein A-I, the Major High Density Lipoprotein Protein, for Site-Specific Oxidation in Human Atherosclerotic Lesions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.337345

    On exposure to the MPO-H 2 O 2 -NaNO 2 system, Tyr-192 is the major nitration target in apoA-I, but Tyr-18 is the major target when apoA-I is associated with HDL. Lipid-free apoA-I (10 μ m ) ( A and B ) or HDL 3 (0.5 mg/ml, ∼12.5 μ m apoA-I)
    Figure Legend Snippet: On exposure to the MPO-H 2 O 2 -NaNO 2 system, Tyr-192 is the major nitration target in apoA-I, but Tyr-18 is the major target when apoA-I is associated with HDL. Lipid-free apoA-I (10 μ m ) ( A and B ) or HDL 3 (0.5 mg/ml, ∼12.5 μ m apoA-I)

    Techniques Used: Nitration

    30) Product Images from "Annotating N Termini for the Human Proteome Project: N Termini and N?-Acetylation Status Differentiate Stable Cleaved Protein Species from Degradation Remnants in the Human Erythrocyte Proteome"

    Article Title: Annotating N Termini for the Human Proteome Project: N Termini and N?-Acetylation Status Differentiate Stable Cleaved Protein Species from Degradation Remnants in the Human Erythrocyte Proteome

    Journal: Journal of Proteome Research

    doi: 10.1021/pr401191w

    Identification of proteins and their termini from erythrocytes. (A) Schematic workflow. Erythrocytes (RBC) were enriched from leukocytes (WBC) by repeated Ficoll density gradient centrifugations, lysed, and separated into membrane, soluble, and soluble hemoglobin-depleted protein fractions. Proteins were denatured and primary amines of proteins with free N-termini and Lys side chains blocked by dimethylation (light gray triangle), followed by digest with trypsin or GluC. Note that unlike shotgun proteomics workflows, TAILS requires labeling at the protein level before trypsin or GluC digestion to isolate and identify the N termini present in the sample. For protein identification, an aliquot of the tryptic digest was removed (preTAILS). N-terminal peptides were enriched using TAILS, including both in vitro dimethylated (gray triangle) and naturally blocked N-termini (black tilted square). Peptides from preTAILS and TAILS were fractionated off-line by SCX-chromatography, analyzed by LC–MS/MS, and identified using three different database search engines before statistical validation and protein identification using the Trans Proteomic Pipeline. (B) Enrichment of erythrocytes and depletion of white blood cells and platelets by repeated gradient (grad.) centrifugations. PLT, platelets; cell counts depicted for: cell pellet, packed cells after serum removal; grad. 1, after first Ficoll gradient; grad. 2, after second Ficoll gradient; final, erythrocyte preparations used to prepare proteome samples. (C) Proteins and (D) N termini identified in membrane (87 MS runs), soluble (147 MS runs), and hemoglobin-depleted (42 MS runs) protein fractions. (E) Proteins and (F) N termini identified in proteome analysis from tryptic digests (preTAILS), enriched N terminal peptides from tryptic digest (Trypsin TAILS) or GluC digest (GluC TAILS). Spectra of (G) trypsin and (H) GluC-digested samples matched to peptide sequences by semispecific database searches with Mascot, X!Tandem, and MS-GF+.
    Figure Legend Snippet: Identification of proteins and their termini from erythrocytes. (A) Schematic workflow. Erythrocytes (RBC) were enriched from leukocytes (WBC) by repeated Ficoll density gradient centrifugations, lysed, and separated into membrane, soluble, and soluble hemoglobin-depleted protein fractions. Proteins were denatured and primary amines of proteins with free N-termini and Lys side chains blocked by dimethylation (light gray triangle), followed by digest with trypsin or GluC. Note that unlike shotgun proteomics workflows, TAILS requires labeling at the protein level before trypsin or GluC digestion to isolate and identify the N termini present in the sample. For protein identification, an aliquot of the tryptic digest was removed (preTAILS). N-terminal peptides were enriched using TAILS, including both in vitro dimethylated (gray triangle) and naturally blocked N-termini (black tilted square). Peptides from preTAILS and TAILS were fractionated off-line by SCX-chromatography, analyzed by LC–MS/MS, and identified using three different database search engines before statistical validation and protein identification using the Trans Proteomic Pipeline. (B) Enrichment of erythrocytes and depletion of white blood cells and platelets by repeated gradient (grad.) centrifugations. PLT, platelets; cell counts depicted for: cell pellet, packed cells after serum removal; grad. 1, after first Ficoll gradient; grad. 2, after second Ficoll gradient; final, erythrocyte preparations used to prepare proteome samples. (C) Proteins and (D) N termini identified in membrane (87 MS runs), soluble (147 MS runs), and hemoglobin-depleted (42 MS runs) protein fractions. (E) Proteins and (F) N termini identified in proteome analysis from tryptic digests (preTAILS), enriched N terminal peptides from tryptic digest (Trypsin TAILS) or GluC digest (GluC TAILS). Spectra of (G) trypsin and (H) GluC-digested samples matched to peptide sequences by semispecific database searches with Mascot, X!Tandem, and MS-GF+.

    Techniques Used: Labeling, In Vitro, Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    31) Product Images from "Evaluation of a Method for Nitrotyrosine Site Identification and Relative Quantitation Using a Stable Isotope-Labeled Nitrated Spike-In Standard and High Resolution Fourier Transform MS and MS/MS Analysis"

    Article Title: Evaluation of a Method for Nitrotyrosine Site Identification and Relative Quantitation Using a Stable Isotope-Labeled Nitrated Spike-In Standard and High Resolution Fourier Transform MS and MS/MS Analysis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms15046265

    ( a ) Base peak ion chromatogram obtained from liquid chromatography (LC)-MS/MS analysis of a microglial cell lysate digest with nitro-BSA mixture (0.25:1 (light:heavy)) added; ( b ) Reconstructed ion chromatogram showing MS/MS spectra that contain the m / z 182 peak with a detection mass tolerance of ±1.0 Da ( m / z 181.0–183.0); and ( c ) Reconstructed ion chromatogram showing MS/MS spectra with high mass accuracy detection of the m / z 182 peak ( m / z 182.056–182.058).
    Figure Legend Snippet: ( a ) Base peak ion chromatogram obtained from liquid chromatography (LC)-MS/MS analysis of a microglial cell lysate digest with nitro-BSA mixture (0.25:1 (light:heavy)) added; ( b ) Reconstructed ion chromatogram showing MS/MS spectra that contain the m / z 182 peak with a detection mass tolerance of ±1.0 Da ( m / z 181.0–183.0); and ( c ) Reconstructed ion chromatogram showing MS/MS spectra with high mass accuracy detection of the m / z 182 peak ( m / z 182.056–182.058).

    Techniques Used: Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    32) Product Images from "Identification of Novel ?-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method"

    Article Title: Identification of Novel ?-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method

    Journal: Neurochemical Research

    doi: 10.1007/s11064-011-0527-x

    Nanoflow LC-ESI-FTICR full scan mass spectra showing the α-syn forms Ac-α-syn 1–139 ( a , b ) and Ac-α-syn 1–140 ( a – c ). Note also the presence of mono- and dioxidized forms. The spectra are summed over a 1.5 min retention time interval around 46 min (50 min gradient). In the spectra the 15 + charge state of the Ac-α-syn 1–139 and Ac-α-syn 1–140 forms are indicated. One SIM scan (3 microscans) of the 15 + ion of the Ac-α-syn 1–103 form ( d , e ). Spectra from the LC–ESI–MS analysis of the Tris fraction of the brain homogenate ( a and d ). Spectra from the LC–ESI–MS analysis of the Triton ® fraction of the brain homogenate ( b and e ). Spectra from the LC–ESI–MS analysis of the SDS fraction of the brain homogenate ( c ). The detected y and b fragments are indicated in the respective sequences ( f ). Cleavage sites labeled with bold lines are confirmed by database search
    Figure Legend Snippet: Nanoflow LC-ESI-FTICR full scan mass spectra showing the α-syn forms Ac-α-syn 1–139 ( a , b ) and Ac-α-syn 1–140 ( a – c ). Note also the presence of mono- and dioxidized forms. The spectra are summed over a 1.5 min retention time interval around 46 min (50 min gradient). In the spectra the 15 + charge state of the Ac-α-syn 1–139 and Ac-α-syn 1–140 forms are indicated. One SIM scan (3 microscans) of the 15 + ion of the Ac-α-syn 1–103 form ( d , e ). Spectra from the LC–ESI–MS analysis of the Tris fraction of the brain homogenate ( a and d ). Spectra from the LC–ESI–MS analysis of the Triton ® fraction of the brain homogenate ( b and e ). Spectra from the LC–ESI–MS analysis of the SDS fraction of the brain homogenate ( c ). The detected y and b fragments are indicated in the respective sequences ( f ). Cleavage sites labeled with bold lines are confirmed by database search

    Techniques Used: Mass Spectrometry, Labeling

    33) Product Images from "Accurate mass and retention time library of serum lipids for type 1 diabetes research"

    Article Title: Accurate mass and retention time library of serum lipids for type 1 diabetes research

    Journal: Analytical and bioanalytical chemistry

    doi: 10.1007/s00216-019-01997-7

    Workflow to create the accurate mass and time tag library for serum lipids. Total lipids were extracted from pooled samples. In the first dimension of LC separation, fractions containing lipid classes from total lipid extraction were separated and collected using mixed-mode LC-ELSD and further analyzed at the molecular level in the second LC dimension using RPLC-MS/MS. Putative identifications obtained from the automated data processing software LipidSearch were manually validated using multiple data filtering criteria (total fatty acid analysis by GC-MS, MS/MS profile, and LC elution order), only the verified lipid identifications were curated into the final lipid library with each species annotated with accurate mass and RPLC retention time
    Figure Legend Snippet: Workflow to create the accurate mass and time tag library for serum lipids. Total lipids were extracted from pooled samples. In the first dimension of LC separation, fractions containing lipid classes from total lipid extraction were separated and collected using mixed-mode LC-ELSD and further analyzed at the molecular level in the second LC dimension using RPLC-MS/MS. Putative identifications obtained from the automated data processing software LipidSearch were manually validated using multiple data filtering criteria (total fatty acid analysis by GC-MS, MS/MS profile, and LC elution order), only the verified lipid identifications were curated into the final lipid library with each species annotated with accurate mass and RPLC retention time

    Techniques Used: Mass Spectrometry, Software, Gas Chromatography-Mass Spectrometry

    34) Product Images from "The siRNA suppressor RTL1 is redox-regulated through glutathionylation of a conserved cysteine in the double-stranded-RNA-binding domain"

    Article Title: The siRNA suppressor RTL1 is redox-regulated through glutathionylation of a conserved cysteine in the double-stranded-RNA-binding domain

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx820

    RTL1 site-specifically cleaves a near-perfect duplex structure in vitro and in planta . ( A ) Left, the hairpin structure predicted from 3′UTR At3g18145 mRNA sequence is shown. The positions of p1 and p2 primers used in primer extension experiments are indicated. The dashed rectangle indicates the sequence corresponding to the RNA substrate-1 shown in ( E ). The sequence corresponding to a 24-nt small RNA is shown by a bar. Arrows show cleavage sites a–d mapped by primer extension. The RNA duplex consensus region rcr1 are boxed ( B ) Primer extension analysis using in vitro transcribed 3′ UTR -At3g18145 and p1 or p2 primers. Arrows (a–d) show mapped cleavage sites in both strands. ( C ) Primer extension analysis on total RNA extracted from Col0 and overexpressing RTL1-Flag #1 plants with p1 primer. Arrows show the rcr1 cleavage positions. ( D ) Cleavage assay of 32 P-CTP RNA substrate-1 using His-RTL1 or His-RTL1m3 proteins. Arrows indicate non cleaved RNA substrate-1 (f0) and cleavage fragment products (f1–f3). A low exposure insert is shown to better visualize the f1 and f2 fragments. DNA size markers are indicated on the left. (E) The predicted 32 P-CTP RNA substrate-1 structure and f1–f3 products are represented.
    Figure Legend Snippet: RTL1 site-specifically cleaves a near-perfect duplex structure in vitro and in planta . ( A ) Left, the hairpin structure predicted from 3′UTR At3g18145 mRNA sequence is shown. The positions of p1 and p2 primers used in primer extension experiments are indicated. The dashed rectangle indicates the sequence corresponding to the RNA substrate-1 shown in ( E ). The sequence corresponding to a 24-nt small RNA is shown by a bar. Arrows show cleavage sites a–d mapped by primer extension. The RNA duplex consensus region rcr1 are boxed ( B ) Primer extension analysis using in vitro transcribed 3′ UTR -At3g18145 and p1 or p2 primers. Arrows (a–d) show mapped cleavage sites in both strands. ( C ) Primer extension analysis on total RNA extracted from Col0 and overexpressing RTL1-Flag #1 plants with p1 primer. Arrows show the rcr1 cleavage positions. ( D ) Cleavage assay of 32 P-CTP RNA substrate-1 using His-RTL1 or His-RTL1m3 proteins. Arrows indicate non cleaved RNA substrate-1 (f0) and cleavage fragment products (f1–f3). A low exposure insert is shown to better visualize the f1 and f2 fragments. DNA size markers are indicated on the left. (E) The predicted 32 P-CTP RNA substrate-1 structure and f1–f3 products are represented.

    Techniques Used: In Vitro, Sequencing, Cleavage Assay

    The native dsRBD and its conserved cysteine C230 are required for RTL1 cleavage of RNA in planta. ( A ) Top, schematic representation of the GU-UG RNA and of the siRNAs produced from the processing of the dsRNA stem by DCL2, DCL3 and DCL4. The GU-UG siRNA construct contains promoter (p35) and terminator (t35S) sequences derived from 35S Cauliflower mosaic virus (CaMV) Bottom, schematic representation of native DCL1, RTL1 and RTL2 and of the truncated proteins DCL1-RD and DCL1-RDD mimicking RTL1 and RTL2, respectively. ( B ) Nicotiana benthamiana leaves co-infiltrated with a 35S:GU-UG construct ( GU-UG ) and either a control 35S-driven construct (GFP), a WT-tagged 35S:RTL1-Myc construct (RTL1) or a mutant 35S:RTL1ΔdsRBD-Myc construct lacking the dsRBD (RTL1ΔdsRBD). Low molecular weight RNAs were extracted and hybridized with a GU probe. Ethidium bromide-stained RNAs are shown as loading control. Proteins were extracted and hybridized with a Myc antibody. Ponceau staining (pink dye) is shown as protein loading control (lanes 2–6). L, indicates protein ladder. ( C ). Left, N. benthamiana leaves co-infiltrated with a 35S:GU-UG construct and either a control 35S-driven construct (GFP), a genomic-based or cDNA-based WT 35S:RTL1 construct (WT genomic and WT cDNA), 35S:RTL1 constructs mutated at cysteine 230 (C230S). Right, western blot analysis show comparable expression of 35S:RTL1 C230), 35S:RTL1 and 35S:RTL1-Myc constructs in N. benthamiana leaves. Proteins were extracted and hybridized with α RTL1 antibody. Ponceau staining (pink dye) is shown as protein loading control (lanes 1–3). ( D ) Nicotiana benthamiana leaves co-infiltrated with a 35S:GU-UG construct and either a control 35S-driven construct (GFP), a genomic-based or cDNA-based WT 35S:RTL1 construct (WT genomic and WT cDNA), 35S:RTL1 constructs mutated at cysteine 230 (C230S) and truncated 35S:DCL1 constructs (DCL1-RDD and DCL1-RD) carrying WT DCL1 sequence (WT) or a mutation at cysteine 1742 (C1742S). Note that RNA samples shown in (C) and d were run on a unique gel, so that the control 35S:GU-UG + 35S:GFP for (D) is visible on (C).
    Figure Legend Snippet: The native dsRBD and its conserved cysteine C230 are required for RTL1 cleavage of RNA in planta. ( A ) Top, schematic representation of the GU-UG RNA and of the siRNAs produced from the processing of the dsRNA stem by DCL2, DCL3 and DCL4. The GU-UG siRNA construct contains promoter (p35) and terminator (t35S) sequences derived from 35S Cauliflower mosaic virus (CaMV) Bottom, schematic representation of native DCL1, RTL1 and RTL2 and of the truncated proteins DCL1-RD and DCL1-RDD mimicking RTL1 and RTL2, respectively. ( B ) Nicotiana benthamiana leaves co-infiltrated with a 35S:GU-UG construct ( GU-UG ) and either a control 35S-driven construct (GFP), a WT-tagged 35S:RTL1-Myc construct (RTL1) or a mutant 35S:RTL1ΔdsRBD-Myc construct lacking the dsRBD (RTL1ΔdsRBD). Low molecular weight RNAs were extracted and hybridized with a GU probe. Ethidium bromide-stained RNAs are shown as loading control. Proteins were extracted and hybridized with a Myc antibody. Ponceau staining (pink dye) is shown as protein loading control (lanes 2–6). L, indicates protein ladder. ( C ). Left, N. benthamiana leaves co-infiltrated with a 35S:GU-UG construct and either a control 35S-driven construct (GFP), a genomic-based or cDNA-based WT 35S:RTL1 construct (WT genomic and WT cDNA), 35S:RTL1 constructs mutated at cysteine 230 (C230S). Right, western blot analysis show comparable expression of 35S:RTL1 C230), 35S:RTL1 and 35S:RTL1-Myc constructs in N. benthamiana leaves. Proteins were extracted and hybridized with α RTL1 antibody. Ponceau staining (pink dye) is shown as protein loading control (lanes 1–3). ( D ) Nicotiana benthamiana leaves co-infiltrated with a 35S:GU-UG construct and either a control 35S-driven construct (GFP), a genomic-based or cDNA-based WT 35S:RTL1 construct (WT genomic and WT cDNA), 35S:RTL1 constructs mutated at cysteine 230 (C230S) and truncated 35S:DCL1 constructs (DCL1-RDD and DCL1-RD) carrying WT DCL1 sequence (WT) or a mutation at cysteine 1742 (C1742S). Note that RNA samples shown in (C) and d were run on a unique gel, so that the control 35S:GU-UG + 35S:GFP for (D) is visible on (C).

    Techniques Used: Produced, Construct, Derivative Assay, Mutagenesis, Molecular Weight, Staining, Western Blot, Expressing, Sequencing

    RTL1 dimerizes through the N-terminal domain. ( A ) Superdex 75 Gel filtration chromatography of His-RTL1 and His-RTL1ΔdsRBD proteins in 150 mM NaCl buffer conditions. The peak positions of conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and ribonuclease A (13.7 kDa) are indicated by arrows ( B ) His-RTL1 protein untreated (lane 1) or treated (lane 2) with DMP was analyzed by western blot using α-RTL1 antibodies ( C ) Coomassie Blue staining of His-RTL1, His-RTL1 C230S or His-RTL1 C260S and His-RTL1ΔdsRBD migrated on native gel in absence of DTT or in the presence of increased amount of DTT. In B and C, arrows indicate positions of monomer (m), dimers (d) and higher order structures (hc) according to standard molecular weight markers.
    Figure Legend Snippet: RTL1 dimerizes through the N-terminal domain. ( A ) Superdex 75 Gel filtration chromatography of His-RTL1 and His-RTL1ΔdsRBD proteins in 150 mM NaCl buffer conditions. The peak positions of conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and ribonuclease A (13.7 kDa) are indicated by arrows ( B ) His-RTL1 protein untreated (lane 1) or treated (lane 2) with DMP was analyzed by western blot using α-RTL1 antibodies ( C ) Coomassie Blue staining of His-RTL1, His-RTL1 C230S or His-RTL1 C260S and His-RTL1ΔdsRBD migrated on native gel in absence of DTT or in the presence of increased amount of DTT. In B and C, arrows indicate positions of monomer (m), dimers (d) and higher order structures (hc) according to standard molecular weight markers.

    Techniques Used: Filtration, Chromatography, Western Blot, Staining, Molecular Weight

    The native dsRBD and conserved cysteine C230 are required for RTL1 cleavage of RNA in vitro . ( A ) Coomassie blue staining and schematic representation of Wt His-RTL1 (R1D1), His-RTL2 (R2D2), swapped His-R1D2 and His-R2D1 or His-RTL1 with mutated cysteines C230S and C260S or truncated HisRTL1ΔdsRBD proteins. Red and yellow boxes correspond to RNase III domains while blue and light blue boxes correspond to dsRBD from RTL1 (R1 and D1) and RTL2 (R2 and D2a and b) proteins respectively. Asterisks show position of mutated Cys 230 and Cys260. ( B ) 32 P-CTP RNA substrate-1 was incubated with 100 ng of His-RTL1(R1D1), His-R1D2, His-R2D1, His-RTL2 (R2D2), His-RTL1C230S, His-RTL1C260S or His-RTL1ΔdsRBD (lane 10) recombinant proteins or with buffer alone. Arrowhead indicates full-length RNA substrate (f0) and major cleavage fragment products (f1 and f2). DNA size markers are indicated on the left. ( C ) Amino acid sequence alignment of dsRBD from RTL1 (F4JK37), RTL2 (Q9LTQ0) and DCL1 (NP_171612), DCL2 (NP_566199), DCL3 (NP_189978) and DCL4 (NP_197532). Vertical arrows heads show highly conserved Cys230 and non-conserved Cys250 and Cys260 in the RTL1 sequence.
    Figure Legend Snippet: The native dsRBD and conserved cysteine C230 are required for RTL1 cleavage of RNA in vitro . ( A ) Coomassie blue staining and schematic representation of Wt His-RTL1 (R1D1), His-RTL2 (R2D2), swapped His-R1D2 and His-R2D1 or His-RTL1 with mutated cysteines C230S and C260S or truncated HisRTL1ΔdsRBD proteins. Red and yellow boxes correspond to RNase III domains while blue and light blue boxes correspond to dsRBD from RTL1 (R1 and D1) and RTL2 (R2 and D2a and b) proteins respectively. Asterisks show position of mutated Cys 230 and Cys260. ( B ) 32 P-CTP RNA substrate-1 was incubated with 100 ng of His-RTL1(R1D1), His-R1D2, His-R2D1, His-RTL2 (R2D2), His-RTL1C230S, His-RTL1C260S or His-RTL1ΔdsRBD (lane 10) recombinant proteins or with buffer alone. Arrowhead indicates full-length RNA substrate (f0) and major cleavage fragment products (f1 and f2). DNA size markers are indicated on the left. ( C ) Amino acid sequence alignment of dsRBD from RTL1 (F4JK37), RTL2 (Q9LTQ0) and DCL1 (NP_171612), DCL2 (NP_566199), DCL3 (NP_189978) and DCL4 (NP_197532). Vertical arrows heads show highly conserved Cys230 and non-conserved Cys250 and Cys260 in the RTL1 sequence.

    Techniques Used: In Vitro, Staining, Incubation, Recombinant, Sequencing

    RNA sequence and structure requirements for RTL1 cleavage. Primer extension analysis using in vitro transcribed 3′UTR RNA (WT and mutated V1-V4) incubated with His-RTL1 protein (+) or buffer alone (−). In the version V1 the A::U and the G::U base pairs within and next to the RNA duplex motif were mutated to G::U and G::C respectively; in V2, three of the A::U base within the RNA duplex motif were mutated to C::G; in V3 the U:: A base pair located in the loop structure was mutated to C::G and in the -V4, the UCG nucleotides in loop structure were mutated to GCU. The Wt and mutated sequences are green and red boxed respectively. Arrow shows cleavage site mapped with p1 primer. Note the two novel cleavage sites (black arrows) adjacent to the a and b sites (black and white arrows) in V1 and the novel cleavage sites adjacent to the a site in V2 and V4. The ΔG (kcal/mol) determined by RNA Mfold for each RNA substrate is indicated.
    Figure Legend Snippet: RNA sequence and structure requirements for RTL1 cleavage. Primer extension analysis using in vitro transcribed 3′UTR RNA (WT and mutated V1-V4) incubated with His-RTL1 protein (+) or buffer alone (−). In the version V1 the A::U and the G::U base pairs within and next to the RNA duplex motif were mutated to G::U and G::C respectively; in V2, three of the A::U base within the RNA duplex motif were mutated to C::G; in V3 the U:: A base pair located in the loop structure was mutated to C::G and in the -V4, the UCG nucleotides in loop structure were mutated to GCU. The Wt and mutated sequences are green and red boxed respectively. Arrow shows cleavage site mapped with p1 primer. Note the two novel cleavage sites (black arrows) adjacent to the a and b sites (black and white arrows) in V1 and the novel cleavage sites adjacent to the a site in V2 and V4. The ΔG (kcal/mol) determined by RNA Mfold for each RNA substrate is indicated.

    Techniques Used: Sequencing, In Vitro, Incubation

    Plant GSSG treatment affects RTL1 cleavage activity. ( A ) Top, proteins from Col0 WT and 35S::RTL1 (#1 and #2) plants were extracted and hybridized with α-Flag and α-RTL1 antibodies. Ponceau staining (pink dye) is shown as protein loading control (lanes 1–3 and 4–6). L, indicates protein ladder. Bottom, sequence of dsRNA substrate-2. The putative rcr motif is underlined and the vertical arrows show DCL3/DCL4 cleavage site. ( B ) [γ- 32 P]-ATP RNA substrate-2 was incubated with either WT (lanes 2), 35S:RTL1-Flag #1 (lane 3) or 35S:RTL1-Flag #2 (lane 5) protein extracts or with WT + 35S:RTL1-Flag #1 (lane 4) or WT + 35S:RTL1-Flag #2 (lane 6) protein extracts. Reactions were performed either with 15 μl, 1.5 μl or 15 μl + 1.5 μl. Lane 1 shows [γ- 32 P]-ATP RNA substrate-2 only. ( C ) Glutathione levels in 2-weeks old WT and 35S:RTL1-Flag #1 plantlets treated or not with 10 mM GSSG. Asterisks indicate a significant difference ( P ≤ 0.01) between total glutathione levels of WT and 35S:RTL1 plants. Reduced and oxidized glutathione levels are indicated by white and gray bars, respectively. The percentage reduction state of glutathione is indicated above bars. Error bars represent SD ( n = 4). ( D ) [γ- 32 P]-ATP RNA substrate-2 was incubated with WT and 35S:RTL1-Flag #1 protein extracts prepared from treated (lanes 3 and 5) or not (lanes 2 and 4) with 10 mM GSSG respectively. Lane 1, shows [γ- 32 P]-ATP RNA substrate-2 only and Lane 6, cleavage reaction using 35S:RTL1-Flag #1 protein extract not submitted to GSSG buffer and is used as a positive control.
    Figure Legend Snippet: Plant GSSG treatment affects RTL1 cleavage activity. ( A ) Top, proteins from Col0 WT and 35S::RTL1 (#1 and #2) plants were extracted and hybridized with α-Flag and α-RTL1 antibodies. Ponceau staining (pink dye) is shown as protein loading control (lanes 1–3 and 4–6). L, indicates protein ladder. Bottom, sequence of dsRNA substrate-2. The putative rcr motif is underlined and the vertical arrows show DCL3/DCL4 cleavage site. ( B ) [γ- 32 P]-ATP RNA substrate-2 was incubated with either WT (lanes 2), 35S:RTL1-Flag #1 (lane 3) or 35S:RTL1-Flag #2 (lane 5) protein extracts or with WT + 35S:RTL1-Flag #1 (lane 4) or WT + 35S:RTL1-Flag #2 (lane 6) protein extracts. Reactions were performed either with 15 μl, 1.5 μl or 15 μl + 1.5 μl. Lane 1 shows [γ- 32 P]-ATP RNA substrate-2 only. ( C ) Glutathione levels in 2-weeks old WT and 35S:RTL1-Flag #1 plantlets treated or not with 10 mM GSSG. Asterisks indicate a significant difference ( P ≤ 0.01) between total glutathione levels of WT and 35S:RTL1 plants. Reduced and oxidized glutathione levels are indicated by white and gray bars, respectively. The percentage reduction state of glutathione is indicated above bars. Error bars represent SD ( n = 4). ( D ) [γ- 32 P]-ATP RNA substrate-2 was incubated with WT and 35S:RTL1-Flag #1 protein extracts prepared from treated (lanes 3 and 5) or not (lanes 2 and 4) with 10 mM GSSG respectively. Lane 1, shows [γ- 32 P]-ATP RNA substrate-2 only and Lane 6, cleavage reaction using 35S:RTL1-Flag #1 protein extract not submitted to GSSG buffer and is used as a positive control.

    Techniques Used: Activity Assay, Staining, Sequencing, Incubation, Positive Control

    In silico and functional analysis of RTL1 suggest a novel regulatory mechanism for RNase III activity in plants. ( A ) Modeled RTL1 (residues 50–284) homodimer based on mouse Dicer (3c4b.1.A) ( 23 ). The RNase III domain of two RTL1 molecules are shown in orange and green while both dsRBD are shown in white. In the left panel, the residues E89, E92, D96 and E185 (E37, E40, D44 and E110 in Aquifex aeolicus RNase III) located in the RNase III domain and required for RNA cleavage are shown. In the right panel, the RTL1 homodimer was rotated 180° and 45° to show conserved cysteines C230 in each dsRBD. The residues T224, N227, E228 and Q231 (T154, Q15, E158 and Q161 in A. aeolicus RNase III) located near to the C230 are indicated. These residues are required for RNA binding of Aa- RNase III. ( B ) In the proposed model, the cysteine C230, which is essential for cleavage activity, is kept in its reduced state (-SH). Glutathionylation of C230 (S-SG) in RTL1 sequence does not affect RTL1 dimerization but it might inhibit dsRNA binding and/or cleavage activity. RTL1 activity inhibition is reversible upon treatment with GRXC1 or GRXC2.
    Figure Legend Snippet: In silico and functional analysis of RTL1 suggest a novel regulatory mechanism for RNase III activity in plants. ( A ) Modeled RTL1 (residues 50–284) homodimer based on mouse Dicer (3c4b.1.A) ( 23 ). The RNase III domain of two RTL1 molecules are shown in orange and green while both dsRBD are shown in white. In the left panel, the residues E89, E92, D96 and E185 (E37, E40, D44 and E110 in Aquifex aeolicus RNase III) located in the RNase III domain and required for RNA cleavage are shown. In the right panel, the RTL1 homodimer was rotated 180° and 45° to show conserved cysteines C230 in each dsRBD. The residues T224, N227, E228 and Q231 (T154, Q15, E158 and Q161 in A. aeolicus RNase III) located near to the C230 are indicated. These residues are required for RNA binding of Aa- RNase III. ( B ) In the proposed model, the cysteine C230, which is essential for cleavage activity, is kept in its reduced state (-SH). Glutathionylation of C230 (S-SG) in RTL1 sequence does not affect RTL1 dimerization but it might inhibit dsRNA binding and/or cleavage activity. RTL1 activity inhibition is reversible upon treatment with GRXC1 or GRXC2.

    Techniques Used: In Silico, Functional Assay, Activity Assay, RNA Binding Assay, Sequencing, Binding Assay, Inhibition

    Glutathionylation of RTL1 affects RNase III cleavage activity. ( A ) His - RTL1 treated with 5 mM GSSG was trypsin digested and analyzed by nanoLC-MSMS. The panels show fragmentation spectra matching peptides with either unmodified (left) or with glutathionylated C230 (right). ( B ) Effect of reduced glutathione (GSSG) on cleavage of the 3′UTR sequence by His-RTL1. The protein samples were incubated at room temperature for 30 min with 0–20 mM GSSG before cleavage assay. ( C ) Effect of oxidized glutathione (GSH) on cleavage of the 3′UTR sequence by His-RTL1 in the presence of H 2 O 2 . The samples were incubated at room temperature for 30 min with 1 mM H 2 O 2 and 0–10 mM GSH. ( D ) Reactivation of cleavage activity His-RTL1 by deglutathionylation with GRXC1. The samples were incubated at room temperature for 30 min with 10 mM GSSG or buffer only. The samples were further incubated at room temperature for 20 min with or without the GRX system (5 mM NADPH, 2 μM GRXC1, 0.45 units GR). Full system is absent in lanes 1–2 while present in lanes 3–4. GR was omitted in lanes 5–6 and no GRXC1 was added in lanes 7–8. Lane 9 is without RTL1 protein. ( E ) Schematic representation of the likely process of deglutathionylation of His-RTL1 by GRX based on a classical deglutathionylation model. The thiolate form of the GRX catalytic Cys attacks the disulfide of the glutathionylated Cys residue, releasing the reduced peptide, and becoming glutathionylated. A molecule of GSH reduces the glutathionylated thiol of GRX, releasing the thiolate catalytic Cys and generating a GSSG molecule. RT-PCR reactions were performed using total RNA from 14 days-old Arabidopsis thaliana plants and primers p1/p9 and U3fw/U3rev .
    Figure Legend Snippet: Glutathionylation of RTL1 affects RNase III cleavage activity. ( A ) His - RTL1 treated with 5 mM GSSG was trypsin digested and analyzed by nanoLC-MSMS. The panels show fragmentation spectra matching peptides with either unmodified (left) or with glutathionylated C230 (right). ( B ) Effect of reduced glutathione (GSSG) on cleavage of the 3′UTR sequence by His-RTL1. The protein samples were incubated at room temperature for 30 min with 0–20 mM GSSG before cleavage assay. ( C ) Effect of oxidized glutathione (GSH) on cleavage of the 3′UTR sequence by His-RTL1 in the presence of H 2 O 2 . The samples were incubated at room temperature for 30 min with 1 mM H 2 O 2 and 0–10 mM GSH. ( D ) Reactivation of cleavage activity His-RTL1 by deglutathionylation with GRXC1. The samples were incubated at room temperature for 30 min with 10 mM GSSG or buffer only. The samples were further incubated at room temperature for 20 min with or without the GRX system (5 mM NADPH, 2 μM GRXC1, 0.45 units GR). Full system is absent in lanes 1–2 while present in lanes 3–4. GR was omitted in lanes 5–6 and no GRXC1 was added in lanes 7–8. Lane 9 is without RTL1 protein. ( E ) Schematic representation of the likely process of deglutathionylation of His-RTL1 by GRX based on a classical deglutathionylation model. The thiolate form of the GRX catalytic Cys attacks the disulfide of the glutathionylated Cys residue, releasing the reduced peptide, and becoming glutathionylated. A molecule of GSH reduces the glutathionylated thiol of GRX, releasing the thiolate catalytic Cys and generating a GSSG molecule. RT-PCR reactions were performed using total RNA from 14 days-old Arabidopsis thaliana plants and primers p1/p9 and U3fw/U3rev .

    Techniques Used: Activity Assay, Sequencing, Incubation, Cleavage Assay, Reverse Transcription Polymerase Chain Reaction

    35) Product Images from "Dissecting ribosomal particles throughout the kingdoms of life using advanced hybrid mass spectrometry methods"

    Article Title: Dissecting ribosomal particles throughout the kingdoms of life using advanced hybrid mass spectrometry methods

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04853-x

    Characterization of ribosomal stalk complexes; composition, post-translational modification and stoichiometry. a Sequence alignment of the flexible tail region of the ribosomal protein L10 from two thermophiles ( T. maritima and T. thermophilus ) and two prokaryotes ( B. subtilis and E. coli ) with chloroplastic L10 from S. oleracea . The presence of three L7/L12 dimer binding sites predicts chloroplast ribosomal stalks to have a heptameric stoichiometry (L10 [L7/L12] 6 ). b Determination of the chloroplastic ribosome stalk stoichiometry using a pseudo-MS3 experiment. Chloroplastic 70S ribosomes are introduced into the gas phase and activated in the source region to release intact stalk complexes (top) with a mass that corresponds well with the predicted heptameric stalks. Isolation and further fragmentation of these complexes releases a single copy of the L12 protein confirming the assignment of the oligomeric state. c A magnificiation of a representative charge state observed in top-down LC-MS/MS analysis of bacterial ( E. coli ) and chloroplastic ( S. oleracea ) ribosomal stalk proteins shows that unlike its bacterial counterpart, chloroplastic ribosomes contain almost no L7 protein and methylation of L7 or L12 is absent. d Human stalk complexes in the 60S ribosome consist of the phosphoproteins P0, P1, and P2. A magnificiation of a representative charge state observed in top-down LC-MS/MS analysis reveals that all three proteins are present in their unphosphorylated (0P), singly phosphorylated (1P), and double phosphorylated forms (2P)
    Figure Legend Snippet: Characterization of ribosomal stalk complexes; composition, post-translational modification and stoichiometry. a Sequence alignment of the flexible tail region of the ribosomal protein L10 from two thermophiles ( T. maritima and T. thermophilus ) and two prokaryotes ( B. subtilis and E. coli ) with chloroplastic L10 from S. oleracea . The presence of three L7/L12 dimer binding sites predicts chloroplast ribosomal stalks to have a heptameric stoichiometry (L10 [L7/L12] 6 ). b Determination of the chloroplastic ribosome stalk stoichiometry using a pseudo-MS3 experiment. Chloroplastic 70S ribosomes are introduced into the gas phase and activated in the source region to release intact stalk complexes (top) with a mass that corresponds well with the predicted heptameric stalks. Isolation and further fragmentation of these complexes releases a single copy of the L12 protein confirming the assignment of the oligomeric state. c A magnificiation of a representative charge state observed in top-down LC-MS/MS analysis of bacterial ( E. coli ) and chloroplastic ( S. oleracea ) ribosomal stalk proteins shows that unlike its bacterial counterpart, chloroplastic ribosomes contain almost no L7 protein and methylation of L7 or L12 is absent. d Human stalk complexes in the 60S ribosome consist of the phosphoproteins P0, P1, and P2. A magnificiation of a representative charge state observed in top-down LC-MS/MS analysis reveals that all three proteins are present in their unphosphorylated (0P), singly phosphorylated (1P), and double phosphorylated forms (2P)

    Techniques Used: Modification, Sequencing, Binding Assay, Isolation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Methylation

    Dissecting ribosomal particles from different organisms and organelles by bottom-up and top-down LC-MS/MS analyses. a – c Relative quantification of both ribosomal and non-ribosomal proteins present in the preparations of So70S, Hs40S, and Hs60S ribosomal particles, respectively. Protein abundance was estimated by using the intensity-based absolute quantification (iBAQ) values of each identified protein. d – f Top-down LC-MS/MS base peak chromatograms of all proteins in the So70S, Hs40S, and Hs60S ribosomal particles, respectively. Top-down LC-MS/MS analysis allows for identification of both ribosomal and non-ribosomal proteins and their distinct proteoforms. g Relative abundance distributions of proteoforms detected in ribosomal proteins of the 40S and 60S subunit identified by database searching. Each dot represents a proteoform of the gene products listed on the x -axis with increasing size representing increasing relative abundance. The position of the dots along the y -axis shows the deviation of measured mass of the proteoform from the mass calculated from the amino acid sequence. Several commonly occurring post-translational modifications (e.g., acetylation, phosphorylation) are annotated with their corresponding mass shift. The data point for L14 is missing since the observed mass shifts introduced by a varying number of inserted alanine repeats is outside the scale of the plot (namely: +213 and +355 Da). To stay consistent, we adopted the ribosomal protein names from Uniprot entries. The pictograms of the Vitruvian man were prepared based on an image that was obtained under a CC0 license from https://commons.wikimedia.org/wiki/File:Digisapiens.png
    Figure Legend Snippet: Dissecting ribosomal particles from different organisms and organelles by bottom-up and top-down LC-MS/MS analyses. a – c Relative quantification of both ribosomal and non-ribosomal proteins present in the preparations of So70S, Hs40S, and Hs60S ribosomal particles, respectively. Protein abundance was estimated by using the intensity-based absolute quantification (iBAQ) values of each identified protein. d – f Top-down LC-MS/MS base peak chromatograms of all proteins in the So70S, Hs40S, and Hs60S ribosomal particles, respectively. Top-down LC-MS/MS analysis allows for identification of both ribosomal and non-ribosomal proteins and their distinct proteoforms. g Relative abundance distributions of proteoforms detected in ribosomal proteins of the 40S and 60S subunit identified by database searching. Each dot represents a proteoform of the gene products listed on the x -axis with increasing size representing increasing relative abundance. The position of the dots along the y -axis shows the deviation of measured mass of the proteoform from the mass calculated from the amino acid sequence. Several commonly occurring post-translational modifications (e.g., acetylation, phosphorylation) are annotated with their corresponding mass shift. The data point for L14 is missing since the observed mass shifts introduced by a varying number of inserted alanine repeats is outside the scale of the plot (namely: +213 and +355 Da). To stay consistent, we adopted the ribosomal protein names from Uniprot entries. The pictograms of the Vitruvian man were prepared based on an image that was obtained under a CC0 license from https://commons.wikimedia.org/wiki/File:Digisapiens.png

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing

    36) Product Images from "NAD+ metabolism governs the proinflammatory senescence-associated secretome"

    Article Title: NAD+ metabolism governs the proinflammatory senescence-associated secretome

    Journal: Nature cell biology

    doi: 10.1038/s41556-019-0287-4

    NAD + metabolism drives proinflammatory SASP. a - c , In established senescent cells, HMGA1 or NAMPT were knocked down using the indicated short hairpin RNAs. The NAMPT activity was also inhibited by FK866. Steady-state metabolite levels were measured by LC-MS/MS. Heat map indicates fold change in comparison to the control condition ( a ) (n=6 independent experiments). NMN ( b ) (n=6 independent experiments) and NAD + /NADH ratio ( c ) were determined in the indicated cells. d , Cells were induced to senesce by oncogenic RAS and analyzed for the NAD + /NADH ratio at the indicated time points. e , Cells with or without ectopic V5-tagged HMGA1 expression with or without NAMPT knockdown were examined for the NAD + /NADH ratio. f , The NAD + /NADH ratio was determined in established senescent cells with or without HMGA1 knockdown with or without ectopic expression of a FLAG-tagged wild type or catalytically-inactive NAMPT. g , h , In established senescence, HMGA1 or NAMPT were knocked down using the indicated shRNAs. The NAMPT activity was also inhibited by FK866. Under these conditions, cells were treated with NMN and the NAD + /NADH ratio ( g ) and expression of the indicated SASP genes ( h ) were determined by qRT-PCR. i - k , Cells from the conditions as in (a) were incubated for 6 hours in the presence of 13 C 6 -glucose and intracellular metabolites were extracted for analysis by LC-MS to evaluate glycolytic flux in the form of pyruvate ( i ) and lactate ( j ) and mitochondrial respiration rates as indicated by citrate production ( k ). Data were normalized based on protein concentration. l , Cells from conditions as in (a) were incubated with a fluorescent glucose analog (2-NBDG) and analyzed by flow cytometry for glucose uptake. m , n , Cells from the conditions in (g) were analyzed using Seahorse Bioanalyzer XF e 96 for extracellular acidification (ECAR) ( m ) and oxygen consumption (OCR) ( n ). Data were normalized based on protein concentration. n = 3 independent experiments unless otherwise stated. All graphs represent mean ± s.d. P values were calculated using a two-tailed t .
    Figure Legend Snippet: NAD + metabolism drives proinflammatory SASP. a - c , In established senescent cells, HMGA1 or NAMPT were knocked down using the indicated short hairpin RNAs. The NAMPT activity was also inhibited by FK866. Steady-state metabolite levels were measured by LC-MS/MS. Heat map indicates fold change in comparison to the control condition ( a ) (n=6 independent experiments). NMN ( b ) (n=6 independent experiments) and NAD + /NADH ratio ( c ) were determined in the indicated cells. d , Cells were induced to senesce by oncogenic RAS and analyzed for the NAD + /NADH ratio at the indicated time points. e , Cells with or without ectopic V5-tagged HMGA1 expression with or without NAMPT knockdown were examined for the NAD + /NADH ratio. f , The NAD + /NADH ratio was determined in established senescent cells with or without HMGA1 knockdown with or without ectopic expression of a FLAG-tagged wild type or catalytically-inactive NAMPT. g , h , In established senescence, HMGA1 or NAMPT were knocked down using the indicated shRNAs. The NAMPT activity was also inhibited by FK866. Under these conditions, cells were treated with NMN and the NAD + /NADH ratio ( g ) and expression of the indicated SASP genes ( h ) were determined by qRT-PCR. i - k , Cells from the conditions as in (a) were incubated for 6 hours in the presence of 13 C 6 -glucose and intracellular metabolites were extracted for analysis by LC-MS to evaluate glycolytic flux in the form of pyruvate ( i ) and lactate ( j ) and mitochondrial respiration rates as indicated by citrate production ( k ). Data were normalized based on protein concentration. l , Cells from conditions as in (a) were incubated with a fluorescent glucose analog (2-NBDG) and analyzed by flow cytometry for glucose uptake. m , n , Cells from the conditions in (g) were analyzed using Seahorse Bioanalyzer XF e 96 for extracellular acidification (ECAR) ( m ) and oxygen consumption (OCR) ( n ). Data were normalized based on protein concentration. n = 3 independent experiments unless otherwise stated. All graphs represent mean ± s.d. P values were calculated using a two-tailed t .

    Techniques Used: Activity Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing, Quantitative RT-PCR, Incubation, Protein Concentration, Flow Cytometry, Cytometry, Two Tailed Test

    37) Product Images from "Hsc70 Is a Novel Interactor of NF-kappaB p65 in Living Hippocampal Neurons"

    Article Title: Hsc70 Is a Novel Interactor of NF-kappaB p65 in Living Hippocampal Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065280

    Hsc70 is a novel neuronal interaction partner of NF-κB. A. Porcine brain extracts were immunoprecipitated with anti NF-κB p65 antibody or isotype control on protein G sepharose in presence of cross-linker. The IP were separated in a 1D SDS gel. Each lane (p65 precipitate and control) were cut into 36 slices and prepared for MS by trypsin digestion. All 36 slices were analyzed by MS. Seven samples in range of 95 to 60 and 27 to 24 kDa were additionally analyzed by LC-ESI-MS/MS. The hits identified by MS included the heat shock cognate Hsc70 as a potential interaction partner of NF-κB p65. B. HEK293 co-transfected with p65-flag and Hsc70-myc or IκBε-myc were lysed followed by co-immunoprecipitation in presence of cross-linker using αmyc (IP) antibody with subsequent WB analysis. A clear interaction band (WB: αFlag) was detectable if myc-tagged IκBε and flag-tagged NF-κB p65 were co-transfected. Similarly, co-transfection of p65-flag and Hsc70-myc resulted in a clear interaction band (WB: αFlag), whereas no band was observed in negative controls (no p65-flag, or no IκBε-myc or Hsc70-myc). Lysates were used as input control. C. Neuronal proteins influence the interaction of NF-κB p65 with Hsc70. IP (αmyc) was performed in presence of cross-linker (DSP) and/or brain lysates with subsequent analysis by western blot. Interaction bands (WB: αFlag) were detectable in cross-linked samples for myc-tagged IκBε and flag tagged NF-κB p65 as well as for Hsc70-myc and NF-κB p65-flag. Combination of cross-linker and brain lysates resulted in stronger interaction band (WB: αFlag) for Hsc70-myc and NF-κB p65-flag. Without cross-linker no interaction bands was detectable.
    Figure Legend Snippet: Hsc70 is a novel neuronal interaction partner of NF-κB. A. Porcine brain extracts were immunoprecipitated with anti NF-κB p65 antibody or isotype control on protein G sepharose in presence of cross-linker. The IP were separated in a 1D SDS gel. Each lane (p65 precipitate and control) were cut into 36 slices and prepared for MS by trypsin digestion. All 36 slices were analyzed by MS. Seven samples in range of 95 to 60 and 27 to 24 kDa were additionally analyzed by LC-ESI-MS/MS. The hits identified by MS included the heat shock cognate Hsc70 as a potential interaction partner of NF-κB p65. B. HEK293 co-transfected with p65-flag and Hsc70-myc or IκBε-myc were lysed followed by co-immunoprecipitation in presence of cross-linker using αmyc (IP) antibody with subsequent WB analysis. A clear interaction band (WB: αFlag) was detectable if myc-tagged IκBε and flag-tagged NF-κB p65 were co-transfected. Similarly, co-transfection of p65-flag and Hsc70-myc resulted in a clear interaction band (WB: αFlag), whereas no band was observed in negative controls (no p65-flag, or no IκBε-myc or Hsc70-myc). Lysates were used as input control. C. Neuronal proteins influence the interaction of NF-κB p65 with Hsc70. IP (αmyc) was performed in presence of cross-linker (DSP) and/or brain lysates with subsequent analysis by western blot. Interaction bands (WB: αFlag) were detectable in cross-linked samples for myc-tagged IκBε and flag tagged NF-κB p65 as well as for Hsc70-myc and NF-κB p65-flag. Combination of cross-linker and brain lysates resulted in stronger interaction band (WB: αFlag) for Hsc70-myc and NF-κB p65-flag. Without cross-linker no interaction bands was detectable.

    Techniques Used: Immunoprecipitation, SDS-Gel, Mass Spectrometry, Transfection, Western Blot, Cotransfection

    38) Product Images from "Alzheimer disease pathology and the cerebrospinal fluid proteome"

    Article Title: Alzheimer disease pathology and the cerebrospinal fluid proteome

    Journal: Alzheimer's Research & Therapy

    doi: 10.1186/s13195-018-0397-4

    Study design and cerebrospinal fluid (CSF) proteome profiling workflow. CSF samples from 120 older individuals with or without cognitive impairment were analyzed using a highly automated shotgun MS-based proteomic workflow. The workflow consists of first removing 14 highly abundant proteins in CSF by immunoaffinity. The rest of the workflow is automated in a 96-well plate format and includes steps of (1) reduction, alkylation, and enzymatic digestion; (2) isobaric labeling and pooling; and (3) purifications. The samples are analyzed with reversed-phase LC-MS/MS, and the data are processed with standard bioinformatic tools
    Figure Legend Snippet: Study design and cerebrospinal fluid (CSF) proteome profiling workflow. CSF samples from 120 older individuals with or without cognitive impairment were analyzed using a highly automated shotgun MS-based proteomic workflow. The workflow consists of first removing 14 highly abundant proteins in CSF by immunoaffinity. The rest of the workflow is automated in a 96-well plate format and includes steps of (1) reduction, alkylation, and enzymatic digestion; (2) isobaric labeling and pooling; and (3) purifications. The samples are analyzed with reversed-phase LC-MS/MS, and the data are processed with standard bioinformatic tools

    Techniques Used: Mass Spectrometry, Labeling, Liquid Chromatography with Mass Spectroscopy

    39) Product Images from "Characterization of Glycosylation Profiles of HIV-1 Transmitted/Founder Envelopes by Mass Spectrometry ▿Characterization of Glycosylation Profiles of HIV-1 Transmitted/Founder Envelopes by Mass Spectrometry ▿ †"

    Article Title: Characterization of Glycosylation Profiles of HIV-1 Transmitted/Founder Envelopes by Mass Spectrometry ▿Characterization of Glycosylation Profiles of HIV-1 Transmitted/Founder Envelopes by Mass Spectrometry ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.05053-11

    (A) LC/ESI-FTICR mass spectrum of the glycopeptide-rich fraction showing three coeluting glycopeptides in the V2-C2, C2, and V4 regions for B.700010040.C9 gp140. (B and C) LC/ESI-MS/MS spectra showing the fragmentation pattern of the glycopeptides in
    Figure Legend Snippet: (A) LC/ESI-FTICR mass spectrum of the glycopeptide-rich fraction showing three coeluting glycopeptides in the V2-C2, C2, and V4 regions for B.700010040.C9 gp140. (B and C) LC/ESI-MS/MS spectra showing the fragmentation pattern of the glycopeptides in

    Techniques Used: Mass Spectrometry

    LC/ESI-FTICR MS data of the doubly glycosylated peptide TIIVHL N 289 ESVNIVCTRPN N 301 NTR in the C.1086 gp140 ΔC sample after trypsin digestion (A), sequential digestion with Endo H and trypsin (B), and sequential digestion with Endo F3 and trypsin
    Figure Legend Snippet: LC/ESI-FTICR MS data of the doubly glycosylated peptide TIIVHL N 289 ESVNIVCTRPN N 301 NTR in the C.1086 gp140 ΔC sample after trypsin digestion (A), sequential digestion with Endo H and trypsin (B), and sequential digestion with Endo F3 and trypsin

    Techniques Used: Mass Spectrometry

    LC/ESI-FTICR mass spectrum of the Env C.1086 gp140 ΔC, which was sequentially digested with Endo H and trypsin. Endo H cleaves high-mannose and hybrid glycans, leaving a single HexNAc on the glycosylated site while complex glycans are unaffected.
    Figure Legend Snippet: LC/ESI-FTICR mass spectrum of the Env C.1086 gp140 ΔC, which was sequentially digested with Endo H and trypsin. Endo H cleaves high-mannose and hybrid glycans, leaving a single HexNAc on the glycosylated site while complex glycans are unaffected.

    Techniques Used:

    LC/ESI-FTICR mass spectra of a glycopeptide-rich fraction showing two coeluting glycopeptides found in the C3 region and V5 loop for the B.700010040.C9 gp140 ΔC Env sample after trypsin digestion (A) and sequential digestion (B) with Endo H and
    Figure Legend Snippet: LC/ESI-FTICR mass spectra of a glycopeptide-rich fraction showing two coeluting glycopeptides found in the C3 region and V5 loop for the B.700010040.C9 gp140 ΔC Env sample after trypsin digestion (A) and sequential digestion (B) with Endo H and

    Techniques Used:

    40) Product Images from "Comparative proteomic analysis of maize (Zea mays L.) seedlings under rice black-streaked dwarf virus infection"

    Article Title: Comparative proteomic analysis of maize (Zea mays L.) seedlings under rice black-streaked dwarf virus infection

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-018-1419-x

    Experimental strategy for quantitative proteome analysis and quality control validation of MS data. a Protein were extracted in three biological replicates for each sample group. All protein samples were trypsin digested and analyzed by HPLC-MS/MS. b Pearson’s correlation of protein quantitation. c Mass delta of all identified peptides. d Length distribution of all identified peptides. 128 label: TMT-128 Label Reagent; and 129-label: TMT-129 Label Reagent (ThermoFisher Scientific, Shanghai, China)
    Figure Legend Snippet: Experimental strategy for quantitative proteome analysis and quality control validation of MS data. a Protein were extracted in three biological replicates for each sample group. All protein samples were trypsin digested and analyzed by HPLC-MS/MS. b Pearson’s correlation of protein quantitation. c Mass delta of all identified peptides. d Length distribution of all identified peptides. 128 label: TMT-128 Label Reagent; and 129-label: TMT-129 Label Reagent (ThermoFisher Scientific, Shanghai, China)

    Techniques Used: Mass Spectrometry, High Performance Liquid Chromatography, Protein Quantitation

    41) Product Images from "Bioavailable Concentrations of Delphinidin and Its Metabolite, Gallic Acid, Induce Antioxidant Protection Associated with Increased Intracellular Glutathione in Cultured Endothelial Cells"

    Article Title: Bioavailable Concentrations of Delphinidin and Its Metabolite, Gallic Acid, Induce Antioxidant Protection Associated with Increased Intracellular Glutathione in Cultured Endothelial Cells

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2017/9260701

    Number of Trypan blue-negative and Trypan blue-positive HUVECs after treatment (24 h) with increasing concentrations of (a) delphinidin (Del) and (b) gallic acid (GA), determined by Trypan blue exclusion. Values are shown as mean ± SEM ( n = 5); ∗ P
    Figure Legend Snippet: Number of Trypan blue-negative and Trypan blue-positive HUVECs after treatment (24 h) with increasing concentrations of (a) delphinidin (Del) and (b) gallic acid (GA), determined by Trypan blue exclusion. Values are shown as mean ± SEM ( n = 5); ∗ P

    Techniques Used:

    Cell viability, measured by MTT, in HUVECs cotreated with pyrogallol (140 μ M) and (a) delphinidin (Del), (b) aged delphinidin (ag Del), or (c) gallic acid (GA). Values are shown as mean ± SEM, ( n = 7); ∗ P
    Figure Legend Snippet: Cell viability, measured by MTT, in HUVECs cotreated with pyrogallol (140 μ M) and (a) delphinidin (Del), (b) aged delphinidin (ag Del), or (c) gallic acid (GA). Values are shown as mean ± SEM, ( n = 7); ∗ P

    Techniques Used: MTT Assay

    (a) Percent of delphinidin (Delph; 200 μ M at T 0 ) remaining in tissue culture medium (pH 7.4, 37°C), when alone and in the presence of the oxidising agent (pyrogallol, 100 μ M) or the reducing agent/antioxidant, ascorbic acid (5 mM). (b) Semilogarithmic representation of percent of Delph remaining in solution; dotted lines indicate the respective half-lives in the presence and absence of ascorbic acid. The results are expressed as mean ± SEM (a) and mean only (b).
    Figure Legend Snippet: (a) Percent of delphinidin (Delph; 200 μ M at T 0 ) remaining in tissue culture medium (pH 7.4, 37°C), when alone and in the presence of the oxidising agent (pyrogallol, 100 μ M) or the reducing agent/antioxidant, ascorbic acid (5 mM). (b) Semilogarithmic representation of percent of Delph remaining in solution; dotted lines indicate the respective half-lives in the presence and absence of ascorbic acid. The results are expressed as mean ± SEM (a) and mean only (b).

    Techniques Used:

    Total GSH concentration (a, b), SOD activity (c, d), and catalase activity (e, f) in HUVECs treated with delphinidin (Del; a, c, and e) and gallic acid (GA; b, d, and f) at concentrations of 1 μ M and 100 nM, measured at 24 h. Values are shown as mean ± SEM ( n = 6); ∗ P
    Figure Legend Snippet: Total GSH concentration (a, b), SOD activity (c, d), and catalase activity (e, f) in HUVECs treated with delphinidin (Del; a, c, and e) and gallic acid (GA; b, d, and f) at concentrations of 1 μ M and 100 nM, measured at 24 h. Values are shown as mean ± SEM ( n = 6); ∗ P

    Techniques Used: Concentration Assay, Activity Assay

    Cell viability, as measured by MTT assay, in HUVECs cotreated with pyocyanin (300 μ M) and (a) delphinidin (Del), (b) aged delphinidin (ag Del), and (c) gallic acid (GA). Values are shown as mean ± SEM ( n = 4); ∗ P
    Figure Legend Snippet: Cell viability, as measured by MTT assay, in HUVECs cotreated with pyocyanin (300 μ M) and (a) delphinidin (Del), (b) aged delphinidin (ag Del), and (c) gallic acid (GA). Values are shown as mean ± SEM ( n = 4); ∗ P

    Techniques Used: MTT Assay

    Effect of pyrogallol alone and (a) pyrogallol + delphinidin (Del) or (b) pyrogallol + gallic acid (GA) on intracellular superoxide in HUVECs. Values are shown as mean ± SEM ( n = 6–8). ∗ P
    Figure Legend Snippet: Effect of pyrogallol alone and (a) pyrogallol + delphinidin (Del) or (b) pyrogallol + gallic acid (GA) on intracellular superoxide in HUVECs. Values are shown as mean ± SEM ( n = 6–8). ∗ P

    Techniques Used:

    LC-MS chromatograms of sample containing (a) standards, (b) culture medium alone, and (c) delphinidin (100 μ M; pH 7.4, temperature 37°C) after 30 minute incubation.
    Figure Legend Snippet: LC-MS chromatograms of sample containing (a) standards, (b) culture medium alone, and (c) delphinidin (100 μ M; pH 7.4, temperature 37°C) after 30 minute incubation.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Incubation

    (a) Ferric reducing ability of plasma (FRAP) of delphinidin (Del) and gallic acid (GA) in culture medium ( n = 3 for each phenolic; ∗∗∗ P
    Figure Legend Snippet: (a) Ferric reducing ability of plasma (FRAP) of delphinidin (Del) and gallic acid (GA) in culture medium ( n = 3 for each phenolic; ∗∗∗ P

    Techniques Used:

    Cell viability, measured by MTT, in HUVECs cotreated with hydrogen peroxide (130 μ M) and (a) delphinidin- (Del-), (b) aged delphinidin- (ag Del-), or (c) gallic acid- (GA-) treated HUVECs. Values are shown as mean ± SEM ( n = 6); ∗∗ P
    Figure Legend Snippet: Cell viability, measured by MTT, in HUVECs cotreated with hydrogen peroxide (130 μ M) and (a) delphinidin- (Del-), (b) aged delphinidin- (ag Del-), or (c) gallic acid- (GA-) treated HUVECs. Values are shown as mean ± SEM ( n = 6); ∗∗ P

    Techniques Used: MTT Assay

    42) Product Images from "Characterization of Early-Phase Neutrophil Extracellular Traps in Urinary Tract Infections"

    Article Title: Characterization of Early-Phase Neutrophil Extracellular Traps in Urinary Tract Infections

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006151

    Proteolytic degradation in extracts of AUP samples. (A) Western Blots were performed with polyclonal antibodies specific for NE, MPO, LTF, and histone H4A. The lane numbers match fraction numbers (UP sol 1, UP sol 3, and UP sol 5) derived from samples #33, #94, and #112. The M r standard consists of ten proteins denoted with kDa values. Green arrowheads point to M r values observed for full-length proteins, including the heavy and light chains of MPO. Full-length NE (29 kDa) and a H4A fragment (8–10 kDa) were detected only in fractions of sample #94. LTF and MPO were represented by full length protein bands in UP sol 3 fractions. The S. aureus protein A (SpA) was detected in a M r range corresponding to its post-translationally modified, cell wall-immobilized forms for sample #112 (red arrowheads). (B) Cleavage sites identified in the peptide sequences of MPO and H4A that were in agreement with the preferred P1 site specificities of the proteases NE and PRTN3. This data is deduced from peptide termini identified via LC-MS/MS from five AUP samples. The N- and C-termini of peptide segments shaded in green include experimentally generated trypsin-specific cleavage sites and PRTN3/NE-specific sites apparently formed as a consequence of the in vivo inflammatory process. Red arrowheads denote sequence positions resulting from protein maturation of precursors. Other arrowheads denote PRTN3 and NE cleavage sites (C-terminal to A, V, L, I, S, T, C, M); if colored black, the site was identified in three or more of the five examined AUP datasets.
    Figure Legend Snippet: Proteolytic degradation in extracts of AUP samples. (A) Western Blots were performed with polyclonal antibodies specific for NE, MPO, LTF, and histone H4A. The lane numbers match fraction numbers (UP sol 1, UP sol 3, and UP sol 5) derived from samples #33, #94, and #112. The M r standard consists of ten proteins denoted with kDa values. Green arrowheads point to M r values observed for full-length proteins, including the heavy and light chains of MPO. Full-length NE (29 kDa) and a H4A fragment (8–10 kDa) were detected only in fractions of sample #94. LTF and MPO were represented by full length protein bands in UP sol 3 fractions. The S. aureus protein A (SpA) was detected in a M r range corresponding to its post-translationally modified, cell wall-immobilized forms for sample #112 (red arrowheads). (B) Cleavage sites identified in the peptide sequences of MPO and H4A that were in agreement with the preferred P1 site specificities of the proteases NE and PRTN3. This data is deduced from peptide termini identified via LC-MS/MS from five AUP samples. The N- and C-termini of peptide segments shaded in green include experimentally generated trypsin-specific cleavage sites and PRTN3/NE-specific sites apparently formed as a consequence of the in vivo inflammatory process. Red arrowheads denote sequence positions resulting from protein maturation of precursors. Other arrowheads denote PRTN3 and NE cleavage sites (C-terminal to A, V, L, I, S, T, C, M); if colored black, the site was identified in three or more of the five examined AUP datasets.

    Techniques Used: Western Blot, Derivative Assay, Modification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Generated, In Vivo, Sequencing

    43) Product Images from "Annotating Nontargeted LC-HRMS/MS Data with Two Complementary Tandem Mass Spectral Libraries"

    Article Title: Annotating Nontargeted LC-HRMS/MS Data with Two Complementary Tandem Mass Spectral Libraries

    Journal: Metabolites

    doi: 10.3390/metabo9010003

    Application of the two tandem mass spectral libraries to the analysis of wastewater samples collected at the WWTP in Innsbruck. Ten influent samples were analyzed. The nontargeted LC-MS/MS data was acquired on a QqTOF instrument using DDA. ( a ) Overview on the number of compounds identified in different compound classes, as well as ( b ) a Venn diagram characterizing the number of identified compounds obtained with the two libraries tested are provided.
    Figure Legend Snippet: Application of the two tandem mass spectral libraries to the analysis of wastewater samples collected at the WWTP in Innsbruck. Ten influent samples were analyzed. The nontargeted LC-MS/MS data was acquired on a QqTOF instrument using DDA. ( a ) Overview on the number of compounds identified in different compound classes, as well as ( b ) a Venn diagram characterizing the number of identified compounds obtained with the two libraries tested are provided.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Application of the two tandem mass spectral libraries to the analysis of wastewater samples collected at the effluent of nine Swiss WWTP. The target and nontargeted LC-MS/MS data was acquired on an Orbitrap instrument using DDA. The column chart visualises the number of identifications obtained with the WRTMD and/or the Eawag library for each sample analyzed.
    Figure Legend Snippet: Application of the two tandem mass spectral libraries to the analysis of wastewater samples collected at the effluent of nine Swiss WWTP. The target and nontargeted LC-MS/MS data was acquired on an Orbitrap instrument using DDA. The column chart visualises the number of identifications obtained with the WRTMD and/or the Eawag library for each sample analyzed.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Application of the two tandem mass spectral libraries to systematic toxicological analysis of 10 authentic plasma samples. Nontargeted LC-MS/MS data was acquired on a QqTOF instrument using DDA. ( a ) Overview on the number of compounds identified in different compound classes via the combined use of the two libraries tested, and ( b ) the Venn diagram illustrating the number of identified compounds obtained with the two libraries tested.
    Figure Legend Snippet: Application of the two tandem mass spectral libraries to systematic toxicological analysis of 10 authentic plasma samples. Nontargeted LC-MS/MS data was acquired on a QqTOF instrument using DDA. ( a ) Overview on the number of compounds identified in different compound classes via the combined use of the two libraries tested, and ( b ) the Venn diagram illustrating the number of identified compounds obtained with the two libraries tested.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    44) Product Images from "Restoring mitochondrial calcium uniporter expression in diabetic mouse heart improves mitochondrial calcium handling and cardiac function"

    Article Title: Restoring mitochondrial calcium uniporter expression in diabetic mouse heart improves mitochondrial calcium handling and cardiac function

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.002066

    Metabolomics analysis confirms the global cardiac-specific effect of AAV-MCU in diabetic mice. A , metabolomics analysis was conducted in plasma and heart, and metabolites that differed significantly were used to generate heat maps. Hierarchical clustering was obtained using Spearman rank correlation. Each row represents one metabolite characterized by specific molecular mass. A color gradient was introduced to visualize relative metabolite levels ( blue = low levels, red = high levels). MCU transgene expression shifted the metabolomics profile toward CTR only in the heart as evidenced by hierarchical clustering. B, glucose levels in heart and plasma samples (glucose is reported in arbitrary units, AU ). All data are presented as mean ± S.D. One-way ANOVA with Tukey's multiple comparisons test was used. *, p
    Figure Legend Snippet: Metabolomics analysis confirms the global cardiac-specific effect of AAV-MCU in diabetic mice. A , metabolomics analysis was conducted in plasma and heart, and metabolites that differed significantly were used to generate heat maps. Hierarchical clustering was obtained using Spearman rank correlation. Each row represents one metabolite characterized by specific molecular mass. A color gradient was introduced to visualize relative metabolite levels ( blue = low levels, red = high levels). MCU transgene expression shifted the metabolomics profile toward CTR only in the heart as evidenced by hierarchical clustering. B, glucose levels in heart and plasma samples (glucose is reported in arbitrary units, AU ). All data are presented as mean ± S.D. One-way ANOVA with Tukey's multiple comparisons test was used. *, p

    Techniques Used: Mouse Assay, Expressing

    45) Product Images from "Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1"

    Article Title: Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1

    Journal: Journal of the American Society for Mass Spectrometry

    doi: 10.1007/s13361-016-1379-z

    The Q-Exactive plus Orbitrap EMR mass spectrometer produces highly resolved spectra which distinguish between the three DJ-1 variants.
    Figure Legend Snippet: The Q-Exactive plus Orbitrap EMR mass spectrometer produces highly resolved spectra which distinguish between the three DJ-1 variants.

    Techniques Used: Mass Spectrometry

    46) Product Images from "Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1"

    Article Title: Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1

    Journal: Journal of the American Society for Mass Spectrometry

    doi: 10.1007/s13361-016-1379-z

    The Q-Exactive plus Orbitrap EMR mass spectrometer produces highly resolved spectra which distinguish between the three DJ-1 variants.
    Figure Legend Snippet: The Q-Exactive plus Orbitrap EMR mass spectrometer produces highly resolved spectra which distinguish between the three DJ-1 variants.

    Techniques Used: Mass Spectrometry

    47) Product Images from "Novel MtCEP1 peptides produced in vivo differentially regulate root development in Medicago truncatula"

    Article Title: Novel MtCEP1 peptides produced in vivo differentially regulate root development in Medicago truncatula

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erv008

    Identification of MtCEP1 peptide species in MtCEP1ox root and vector control exudates. (A) The pre-propeptide structure of MtCEP1 showing the two 15 amino acid peptide domains. (B) Eight species of MtCEP1 domain 1 (D1) peptide and one species of the domain 2 (D2) peptide were identified with their respective PTMs. HyP: hydroxylated proline; TaP: tri-arabinosylated proline. (C) The relative concentration of the nine MtCEP1 peptide species found in MtCEP1ox exudate and the five species found in the vector control exudate. Serial dilutions of the synthetic peptide were performed to establish a standard calibration curve (20amol to 200 femtomol) from which the concentration of each peptide was extrapolated. (D) The peptide species in MtCEP1ox samples were analysed using Quadrupole-Orbitrap and Q-TOF mass spectrometers. The ratio of the five peptide species identified correlated well between both nano-LC-ESI-MS systems. (E) The five most abundant peptides in MtCEP1ox sample eluted from the column based on their hydrophobicity as indicated by their retention time in the Quadrapole-Orbitrap. (F) The peptides in the vector control samples were detected in relative minute amounts as shown in the extracted ion chromatogram.
    Figure Legend Snippet: Identification of MtCEP1 peptide species in MtCEP1ox root and vector control exudates. (A) The pre-propeptide structure of MtCEP1 showing the two 15 amino acid peptide domains. (B) Eight species of MtCEP1 domain 1 (D1) peptide and one species of the domain 2 (D2) peptide were identified with their respective PTMs. HyP: hydroxylated proline; TaP: tri-arabinosylated proline. (C) The relative concentration of the nine MtCEP1 peptide species found in MtCEP1ox exudate and the five species found in the vector control exudate. Serial dilutions of the synthetic peptide were performed to establish a standard calibration curve (20amol to 200 femtomol) from which the concentration of each peptide was extrapolated. (D) The peptide species in MtCEP1ox samples were analysed using Quadrupole-Orbitrap and Q-TOF mass spectrometers. The ratio of the five peptide species identified correlated well between both nano-LC-ESI-MS systems. (E) The five most abundant peptides in MtCEP1ox sample eluted from the column based on their hydrophobicity as indicated by their retention time in the Quadrapole-Orbitrap. (F) The peptides in the vector control samples were detected in relative minute amounts as shown in the extracted ion chromatogram.

    Techniques Used: Plasmid Preparation, Concentration Assay, Mass Spectrometry

    48) Product Images from "BAP1 induces cell death via interaction with 14-3-3 in neuroblastoma"

    Article Title: BAP1 induces cell death via interaction with 14-3-3 in neuroblastoma

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0500-6

    Identification of 14-3-3 protein as a novel binding partner to BAP1. a Representative LC–MS/MS chromatogram of in-gel digested sample depicted in red, an example of the peptide from 1433S protein eluted at 70.8 min. The Orbitrap mass spectra of the quadruply charged 1433S peptide sequence 191–224 (m/z 836.9045) enabled mass determination of the peptide (isotopic distribution depicted in gray). The assigned fragmentation pattern of sequence 191–224 peptide is given in black. b , c SK-N-RA cells were transfected with FLAG-tagged full-length BAP1 or Flag-empty vector (Ctrl) and lysates were immunoprecipitated with antibodies against FLAG ( b ) or 14-3-3 ( c ) and probed with FLAG to detect BAP1 or 14-3-3 as indicated in the figure ( n = 3). d TR-FRET assay using FRET buffer (Ctrl), purified control GST (negative control), GST-14-3-3 protein, and His-BAP1 mixed in FRET buffer, followed by addition of anti-GST-Terbium and anti-His-D2 antibodies. The FRET signals were detected using an Envision spectrophotometer. The data were presented as the ratio of counts at 665/counts at 620 nm × 10 4 with standard deviation calculated from triplicate samples (mean ± s.e.m., n = 3, * P
    Figure Legend Snippet: Identification of 14-3-3 protein as a novel binding partner to BAP1. a Representative LC–MS/MS chromatogram of in-gel digested sample depicted in red, an example of the peptide from 1433S protein eluted at 70.8 min. The Orbitrap mass spectra of the quadruply charged 1433S peptide sequence 191–224 (m/z 836.9045) enabled mass determination of the peptide (isotopic distribution depicted in gray). The assigned fragmentation pattern of sequence 191–224 peptide is given in black. b , c SK-N-RA cells were transfected with FLAG-tagged full-length BAP1 or Flag-empty vector (Ctrl) and lysates were immunoprecipitated with antibodies against FLAG ( b ) or 14-3-3 ( c ) and probed with FLAG to detect BAP1 or 14-3-3 as indicated in the figure ( n = 3). d TR-FRET assay using FRET buffer (Ctrl), purified control GST (negative control), GST-14-3-3 protein, and His-BAP1 mixed in FRET buffer, followed by addition of anti-GST-Terbium and anti-His-D2 antibodies. The FRET signals were detected using an Envision spectrophotometer. The data were presented as the ratio of counts at 665/counts at 620 nm × 10 4 with standard deviation calculated from triplicate samples (mean ± s.e.m., n = 3, * P

    Techniques Used: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Transfection, Plasmid Preparation, Immunoprecipitation, Purification, Negative Control, Spectrophotometry, Standard Deviation

    49) Product Images from "Genotype-Specific Modulatory Effects of Select Spectral Bandwidths on the Nutritive and Phytochemical Composition of Microgreens"

    Article Title: Genotype-Specific Modulatory Effects of Select Spectral Bandwidths on the Nutritive and Phytochemical Composition of Microgreens

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2019.01501

    Principal component loading plot and scores of principal component analysis (PCA) of the concentrations of 13 key phenolic compounds and total polyphenols identified and quantitated by UHPLC-Q-Orbitrap HRMS analysis in four microgreens genotypes as modulated by variable spectral bandwidths in a controlled growth environment.
    Figure Legend Snippet: Principal component loading plot and scores of principal component analysis (PCA) of the concentrations of 13 key phenolic compounds and total polyphenols identified and quantitated by UHPLC-Q-Orbitrap HRMS analysis in four microgreens genotypes as modulated by variable spectral bandwidths in a controlled growth environment.

    Techniques Used:

    50) Product Images from "Metabolomics meets functional assays: coupling LC–MS and microfluidic cell-based receptor-ligand analyses"

    Article Title: Metabolomics meets functional assays: coupling LC–MS and microfluidic cell-based receptor-ligand analyses

    Journal: Metabolomics

    doi: 10.1007/s11306-016-1057-y

    Scheme of an online functional metabolomics setup. The microfluidic flowcell with sensory cells is placed in a microscopic fluorescence detector, which will continuously monitor the cellular response of sample compounds eluting from the LC column. Simultaneous detection of compounds by UV–Vis ( lower chromatogram ) and MS ( upper chromatogram ) can provide identification of bioactive compounds. LC-fractions, preferably separated into individual compounds, are collected as well in order to keep a compound record of the run when further functional analysis or metabolite identification experiments are required. This figure is modified from www.galenica.cl
    Figure Legend Snippet: Scheme of an online functional metabolomics setup. The microfluidic flowcell with sensory cells is placed in a microscopic fluorescence detector, which will continuously monitor the cellular response of sample compounds eluting from the LC column. Simultaneous detection of compounds by UV–Vis ( lower chromatogram ) and MS ( upper chromatogram ) can provide identification of bioactive compounds. LC-fractions, preferably separated into individual compounds, are collected as well in order to keep a compound record of the run when further functional analysis or metabolite identification experiments are required. This figure is modified from www.galenica.cl

    Techniques Used: Functional Assay, Fluorescence, Mass Spectrometry, Modification

    51) Product Images from "Identification of Potential Sites for Tryptophan Oxidation in Recombinant Antibodies Using tert-Butylhydroperoxide and Quantitative LC-MS"

    Article Title: Identification of Potential Sites for Tryptophan Oxidation in Recombinant Antibodies Using tert-Butylhydroperoxide and Quantitative LC-MS

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017708

    LC-separation UV-profiles of the real-time stability sample (9 months at 4°C) and the corresponding reference material (RS, stored at −70°C) after tryptic cleavage. The m/z -values of Met-253 and Trp-32 containing tryptic antibody peptides, obtained by LC-ESI-MS, are listed in the inset along with their corresponding LC fraction number. Peak identification and quantification performed by LC-MS. Chromatographic conditions are described in the materials and methods .
    Figure Legend Snippet: LC-separation UV-profiles of the real-time stability sample (9 months at 4°C) and the corresponding reference material (RS, stored at −70°C) after tryptic cleavage. The m/z -values of Met-253 and Trp-32 containing tryptic antibody peptides, obtained by LC-ESI-MS, are listed in the inset along with their corresponding LC fraction number. Peak identification and quantification performed by LC-MS. Chromatographic conditions are described in the materials and methods .

    Techniques Used: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    52) Product Images from "An integrated workflow for characterizing intact phosphoproteins from complex mixtures"

    Article Title: An integrated workflow for characterizing intact phosphoproteins from complex mixtures

    Journal: Analytical chemistry

    doi: 10.1021/ac802487q

    Integrated analysis of a standard phosphoprotein mixture. “Bird’s eye view” offered by LC/MS data facilitates relative quantitation of α2-casein (a), α1- casein (b), and β-casein (c) phosphoprotein isoforms.
    Figure Legend Snippet: Integrated analysis of a standard phosphoprotein mixture. “Bird’s eye view” offered by LC/MS data facilitates relative quantitation of α2-casein (a), α1- casein (b), and β-casein (c) phosphoprotein isoforms.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Quantitation Assay

    53) Product Images from "SILAC-Based Quantitative Proteomic Analysis Unveils Arsenite-Induced Perturbation of Multiple Pathways in Human Skin Fibroblast Cells"

    Article Title: SILAC-Based Quantitative Proteomic Analysis Unveils Arsenite-Induced Perturbation of Multiple Pathways in Human Skin Fibroblast Cells

    Journal: Chemical research in toxicology

    doi: 10.1021/acs.chemrestox.6b00416

    Forward- and reverse-SILAC combined with LC/MS/MS for the comparative analysis of protein expression in GM00637 cells upon arsenite treatment (A). Pie chart displaying the distribution of expression ratios (treated/untreated) for the quantified proteins (B) and Venn diagram revealing the number of quantified proteins (C) from three independent experiments.
    Figure Legend Snippet: Forward- and reverse-SILAC combined with LC/MS/MS for the comparative analysis of protein expression in GM00637 cells upon arsenite treatment (A). Pie chart displaying the distribution of expression ratios (treated/untreated) for the quantified proteins (B) and Venn diagram revealing the number of quantified proteins (C) from three independent experiments.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing

    54) Product Images from "FVIIa-sTF and Thrombin Inhibitory Activities of Compounds Isolated from Microcystis aeruginosa K-139"

    Article Title: FVIIa-sTF and Thrombin Inhibitory Activities of Compounds Isolated from Microcystis aeruginosa K-139

    Journal: Marine Drugs

    doi: 10.3390/md15090275

    Advanced Marfey analysis of Leu in aeruginosin K139 using LC-MS in 25% acetonitrile with 0.1% formic acid to 65% acetonitrile with 0.1% formic acid over 45 min, TSKgel SuperODS (TOSOH) 100 × 2.0 mm, capillary temp 250 °C, 25 µL injection of 1 mg/mL. ( A ). Extracted Ion Chromatogram (EIC) with m/z 426–427 of dl- Leu- l- FDLA; ( B ). EIC of l -Leu- l -FDLA; ( C ). EIC of aeruginosin K139- l -FDLA; ( D ). m/z of dl- Leu- l -FDLA with a retention time (t R , min) 12.1; ( E ). m/z of dl -Leu -l -FDLA with a retention time (t R , min) 20.3; ( F ). m/z of l -Leu- l -FDLA with a retention time (t R , min) 12.2; ( G ). m/z of aeruginosin K139- l- FDLA with a retention time (t R , min) 12.5.
    Figure Legend Snippet: Advanced Marfey analysis of Leu in aeruginosin K139 using LC-MS in 25% acetonitrile with 0.1% formic acid to 65% acetonitrile with 0.1% formic acid over 45 min, TSKgel SuperODS (TOSOH) 100 × 2.0 mm, capillary temp 250 °C, 25 µL injection of 1 mg/mL. ( A ). Extracted Ion Chromatogram (EIC) with m/z 426–427 of dl- Leu- l- FDLA; ( B ). EIC of l -Leu- l -FDLA; ( C ). EIC of aeruginosin K139- l -FDLA; ( D ). m/z of dl- Leu- l -FDLA with a retention time (t R , min) 12.1; ( E ). m/z of dl -Leu -l -FDLA with a retention time (t R , min) 20.3; ( F ). m/z of l -Leu- l -FDLA with a retention time (t R , min) 12.2; ( G ). m/z of aeruginosin K139- l- FDLA with a retention time (t R , min) 12.5.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Injection

    55) Product Images from "Simple enrichment and analysis of plasma lysophosphatidic acids"

    Article Title: Simple enrichment and analysis of plasma lysophosphatidic acids

    Journal: The Analyst

    doi: 10.1039/c3an01168b

    LC-ESI/MS/MS traces of a 10 μM standard mixture of LPAs. Column: Luna ™ C-8 (50 × 2 mm, 3 μm) at 40 °C. Injection volume: 10 μL. Mobile phase: 9:1 MeOH:aqueous HCOOH (pH 2.5) at a flow rate of 0.4 mL/min.
    Figure Legend Snippet: LC-ESI/MS/MS traces of a 10 μM standard mixture of LPAs. Column: Luna ™ C-8 (50 × 2 mm, 3 μm) at 40 °C. Injection volume: 10 μL. Mobile phase: 9:1 MeOH:aqueous HCOOH (pH 2.5) at a flow rate of 0.4 mL/min.

    Techniques Used: Mass Spectrometry, Injection, Flow Cytometry

    Calibration curves of LPAs using the LC-ESI/MS/MS method. The area ratio is the peak area of individual LPAs divided by the peak area of the internal standard (LPA 17:0).
    Figure Legend Snippet: Calibration curves of LPAs using the LC-ESI/MS/MS method. The area ratio is the peak area of individual LPAs divided by the peak area of the internal standard (LPA 17:0).

    Techniques Used: Mass Spectrometry

    56) Product Images from "Monitoring of the spatial and temporal dynamics of BER/SSBR pathway proteins, including MYH, UNG2, MPG, NTH1 and NEIL1-3, during DNA replication"

    Article Title: Monitoring of the spatial and temporal dynamics of BER/SSBR pathway proteins, including MYH, UNG2, MPG, NTH1 and NEIL1-3, during DNA replication

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx476

    Replication and BER/SSBR proteins detected by iPOND-PRM. Bars represent normalized peptide peak areas of replisome enriched (light gray), chromatin enriched (dark gray) and proteins with no enrichment (mid gray). Mean ± SD are shown for PCNA, POLδ, POLϵ, FEN1, LIG1, LIG3, XRCC1, APE1, MPG, NEIL1, NTH1 (all n = 5), NEIL3 ( n = 4) and MYH ( n = 3). For UNG2, POLβ and NEIL2, each biological replicate is illustrated. Statistical significance was calculated using a two-sided paired Student's t -test, where *: P
    Figure Legend Snippet: Replication and BER/SSBR proteins detected by iPOND-PRM. Bars represent normalized peptide peak areas of replisome enriched (light gray), chromatin enriched (dark gray) and proteins with no enrichment (mid gray). Mean ± SD are shown for PCNA, POLδ, POLϵ, FEN1, LIG1, LIG3, XRCC1, APE1, MPG, NEIL1, NTH1 (all n = 5), NEIL3 ( n = 4) and MYH ( n = 3). For UNG2, POLβ and NEIL2, each biological replicate is illustrated. Statistical significance was calculated using a two-sided paired Student's t -test, where *: P

    Techniques Used:

    57) Product Images from "Time-scale dynamics of proteome and transcriptome of the white-rot fungus Phlebia radiata: growth on spruce wood and decay effect on lignocellulose"

    Article Title: Time-scale dynamics of proteome and transcriptome of the white-rot fungus Phlebia radiata: growth on spruce wood and decay effect on lignocellulose

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-016-0608-9

    Functional distribution of proteins identified in P. radiata proteome on spruce wood by LC–MS/MS peptide analysis. a Distribution of the total identified proteins (1356) into functional classes. b Venn diagram of distribution of identified proteins (1349) in the fungal proteomes on wood extracted from five weekly time points. c Distribution in percentage of wood-decay CAZy and other proteins at the six time points. Proteins with equal or more than two unique peptides were included in the analyses
    Figure Legend Snippet: Functional distribution of proteins identified in P. radiata proteome on spruce wood by LC–MS/MS peptide analysis. a Distribution of the total identified proteins (1356) into functional classes. b Venn diagram of distribution of identified proteins (1349) in the fungal proteomes on wood extracted from five weekly time points. c Distribution in percentage of wood-decay CAZy and other proteins at the six time points. Proteins with equal or more than two unique peptides were included in the analyses

    Techniques Used: Functional Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    58) Product Images from "Measurement of endogenous versus exogenous formaldehyde-induced DNA-protein crosslinks in animal tissues by stable isotope labeling and ultrasensitive mass spectrometry"

    Article Title: Measurement of endogenous versus exogenous formaldehyde-induced DNA-protein crosslinks in animal tissues by stable isotope labeling and ultrasensitive mass spectrometry

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-15-2527

    Typical Nano-LC-ESI-MS/MS-SRM chromatogram and calibration curve of dG-Me-Cys. Nano-LC-ESI-MS/MS-SRM chromatograms of dG-Me-Cys (0.0375 fmol) and Internal Standard [ 15 N 5 ]-dG-[ 13 CD 2 ]-Me-Cys (0.5 fmol). NL = normalized spectrum to largest peak in particular
    Figure Legend Snippet: Typical Nano-LC-ESI-MS/MS-SRM chromatogram and calibration curve of dG-Me-Cys. Nano-LC-ESI-MS/MS-SRM chromatograms of dG-Me-Cys (0.0375 fmol) and Internal Standard [ 15 N 5 ]-dG-[ 13 CD 2 ]-Me-Cys (0.5 fmol). NL = normalized spectrum to largest peak in particular

    Techniques Used: Mass Spectrometry

    59) Product Images from "Cellular Interactome Dynamics during Paclitaxel Treatment"

    Article Title: Cellular Interactome Dynamics during Paclitaxel Treatment

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.10.063

    Experimental Overview (A) Cells are cultured in SILAC media; the isotopically light or heavy cells are treated with the mitotic inhibitors PTX (5, 10, 20, 50, 100, and 500 nM), NOC (3 μM), CA4 (5 nM), and STLC (5 μM); and the corresponding isotope pair is treated with 0.1% (v/v) DMSO vehicle control. (B) Cells are cross-linked with 10 mM BDP-NHP, followed by lysis and protein extraction with 8 M urea and tryptic digestion. (C) Peptide samples are fractionated by strong cation exchange (SCX) chromatography, with early eluting fractions (1–5) containing non-cross-linked peptides used for global proteome quantification and later fractions(6–14) subjected to avidin affinity chromatography and used for cross-linked peptide pair identification and quantification. (D) LC-MS analysis consists of MS1 measurement of light and heavy SILAC isotope precursor ions followed by MS2 analysis of fragment ions. For cross-linked peptide pairs, detection of a protein interaction reporter (PIR) mass relationship triggers MS3 of the released peptides for sequence determination. (E) Data analysis consists of integrating protein-level and cross-link-level quantitative information. Drug concentration-dependent trends in cross-linked peptide pairs are identified by statistical filtering and longitudinal k-means clustering. Cross-link data are analyzed in terms of protein structural information. (F) A collection of nine representative images of HeLa cells treated with PTX. PTX concentration increases (0, 20, and 50 nM) while scanning the images from the top to bottom, and treatment time increases(0, 3, and 18 h) from left to right. The cells are colored yellow by a software-applied mask, while rounded cells that are locked in mitosis are colored magenta. Scale bar of 1000 μm indicated on lower right image. (G) Line graph plotting the fraction of mitotic cells on the y axis and PTX treatment time on the x axis (biological replicates, n = 3).
    Figure Legend Snippet: Experimental Overview (A) Cells are cultured in SILAC media; the isotopically light or heavy cells are treated with the mitotic inhibitors PTX (5, 10, 20, 50, 100, and 500 nM), NOC (3 μM), CA4 (5 nM), and STLC (5 μM); and the corresponding isotope pair is treated with 0.1% (v/v) DMSO vehicle control. (B) Cells are cross-linked with 10 mM BDP-NHP, followed by lysis and protein extraction with 8 M urea and tryptic digestion. (C) Peptide samples are fractionated by strong cation exchange (SCX) chromatography, with early eluting fractions (1–5) containing non-cross-linked peptides used for global proteome quantification and later fractions(6–14) subjected to avidin affinity chromatography and used for cross-linked peptide pair identification and quantification. (D) LC-MS analysis consists of MS1 measurement of light and heavy SILAC isotope precursor ions followed by MS2 analysis of fragment ions. For cross-linked peptide pairs, detection of a protein interaction reporter (PIR) mass relationship triggers MS3 of the released peptides for sequence determination. (E) Data analysis consists of integrating protein-level and cross-link-level quantitative information. Drug concentration-dependent trends in cross-linked peptide pairs are identified by statistical filtering and longitudinal k-means clustering. Cross-link data are analyzed in terms of protein structural information. (F) A collection of nine representative images of HeLa cells treated with PTX. PTX concentration increases (0, 20, and 50 nM) while scanning the images from the top to bottom, and treatment time increases(0, 3, and 18 h) from left to right. The cells are colored yellow by a software-applied mask, while rounded cells that are locked in mitosis are colored magenta. Scale bar of 1000 μm indicated on lower right image. (G) Line graph plotting the fraction of mitotic cells on the y axis and PTX treatment time on the x axis (biological replicates, n = 3).

    Techniques Used: Cell Culture, Lysis, Protein Extraction, Chromatography, Avidin-Biotin Assay, Affinity Chromatography, Liquid Chromatography with Mass Spectroscopy, Sequencing, Concentration Assay, Software

    60) Product Images from "Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport"

    Article Title: Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201702137

    Proteomic quantification of the SNX-BAR interactome identifies the IGF1R as a SNX-BAR interactor. (A) GFP-tagged SNX1, SNX2, SNX5, and SNX6 were transiently expressed in SILAC-labeled HEK293 cells. GFP-only was expressed in nonlabeled HEK293 cells as a control. The SNX-BARs and GFP were precipitated with GFP-trap beads, each SNX-BAR IP was pooled with a GFP-only IP, and the combined samples were quantified by LC-MS/MS. The panel displays the fold enrichment of the other SNX-BARs in each individual SNX-BAR precipitation, indicating robust heterodimerization and multimerization between the SNX-BARs. Selected other interactors of the respective SNX-BAR protein at least fivefold enriched over the GFP-only control are indicated by arrows. (B) GFP-trap IPs of the indicated SNX-BARs and analysis of the presence of the endogenous IGF1R and the INSR in the precipitates. (C) GFP-trap IPs of the isolated cytoplasmic tails of selected potential SNX-BAR cargoes and analysis of the presence of the endogenous SNX-BAR protein in the precipitates. IB, immunoblot. (D) Direct recombinant interaction with the GST-tagged cytosolic tails produced in bacteria and His-tagged SNX5, and SNX6 coexpressed with His-tagged SNX1 and SNX2, respectively. (E) Immunofluorescent staining of endogenous IGF1R (green) and endogenous SNX1 (red) in serum-starved MCF-7 cells treated with 10 nM IGF-1 for the indicated periods. (F) Degradation assay of endogenous IGF1R and the INSR in HeLa cells, and clonal SNX1/2 and SNX5/6 double-KO cells lines treated with the ribosomal inhibitor cycloheximide for the indicated periods. The degradation kinetics were quantified over four independent experiments. Bars, 10 µm. Error bars indicate SD. *, P
    Figure Legend Snippet: Proteomic quantification of the SNX-BAR interactome identifies the IGF1R as a SNX-BAR interactor. (A) GFP-tagged SNX1, SNX2, SNX5, and SNX6 were transiently expressed in SILAC-labeled HEK293 cells. GFP-only was expressed in nonlabeled HEK293 cells as a control. The SNX-BARs and GFP were precipitated with GFP-trap beads, each SNX-BAR IP was pooled with a GFP-only IP, and the combined samples were quantified by LC-MS/MS. The panel displays the fold enrichment of the other SNX-BARs in each individual SNX-BAR precipitation, indicating robust heterodimerization and multimerization between the SNX-BARs. Selected other interactors of the respective SNX-BAR protein at least fivefold enriched over the GFP-only control are indicated by arrows. (B) GFP-trap IPs of the indicated SNX-BARs and analysis of the presence of the endogenous IGF1R and the INSR in the precipitates. (C) GFP-trap IPs of the isolated cytoplasmic tails of selected potential SNX-BAR cargoes and analysis of the presence of the endogenous SNX-BAR protein in the precipitates. IB, immunoblot. (D) Direct recombinant interaction with the GST-tagged cytosolic tails produced in bacteria and His-tagged SNX5, and SNX6 coexpressed with His-tagged SNX1 and SNX2, respectively. (E) Immunofluorescent staining of endogenous IGF1R (green) and endogenous SNX1 (red) in serum-starved MCF-7 cells treated with 10 nM IGF-1 for the indicated periods. (F) Degradation assay of endogenous IGF1R and the INSR in HeLa cells, and clonal SNX1/2 and SNX5/6 double-KO cells lines treated with the ribosomal inhibitor cycloheximide for the indicated periods. The degradation kinetics were quantified over four independent experiments. Bars, 10 µm. Error bars indicate SD. *, P

    Techniques Used: Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Isolation, Recombinant, Produced, Staining, Degradation Assay

    SNX-BAR dimers directly bind to the CI-MPR tail to promote retrograde sorting to the TGN. (A) The GFP-tagged CI-MPR tail and GFP only were lentivirally expressed in SILAC-labeled HEK293 cells and precipitated with GFP-trap beads, and then interacting proteins were identified by LC-MS/MS. The panel shows a STRING network analysis of CI-MPR tail–interacting proteins. The numbers beneath each protein indicate the fold enrichment of the protein when compared with the GFP-only control. (B) Western blot–based verification of the interacting proteins. (C) GFP-trap IPs of the indicated GFP-tagged SNX-BAR proteins precipitating the endogenous CI-MPR from HEK293 cells. (D) GFP-trap IPs of the GFP-tagged WT CI-MPR tail and of the indicated mutant CI-MPR tail constructs were probed for the presence of the indicated endogenous SNXs and endogenous VPS35 by Western blotting. (E) Direct recombinant interaction assay with GST-tagged CI-MPR tail produced in bacteria and the indicated individual His-tagged SNX-BAR proteins produced as secreted proteins in HEK293 cells. (F) Direct recombinant interaction assay with GST-tagged CI-MPR tail produced in bacteria, and the indicated His-tagged SNX-BAR proteins coexpressed as secreted proteins in HEK293 cells. (G) Three CRISPR constructs per indicated gene targeting distinct genomic regions of the respective gene were pooled and cotransfected with a puromycin resistance–encoding plasmid. After puromycin selection and recovery for 3 d, cells were seeded onto coverslips, and CI-MPR localization to the TGN was analyzed by immunofluorescent staining of endogenous CI-MPR (red) and endogenous TGN46 (green). The screen was performed twice, and the colocalization (coloc) between CI-MPR and TGN46 was quantified over 20 images per condition acquired in the two experiments. The efficiency of the respective CRISPR-Cas9 treatment was verified by Western blotting against selected targets. Bars, 10 µm. Error bars indicate SD. *, P
    Figure Legend Snippet: SNX-BAR dimers directly bind to the CI-MPR tail to promote retrograde sorting to the TGN. (A) The GFP-tagged CI-MPR tail and GFP only were lentivirally expressed in SILAC-labeled HEK293 cells and precipitated with GFP-trap beads, and then interacting proteins were identified by LC-MS/MS. The panel shows a STRING network analysis of CI-MPR tail–interacting proteins. The numbers beneath each protein indicate the fold enrichment of the protein when compared with the GFP-only control. (B) Western blot–based verification of the interacting proteins. (C) GFP-trap IPs of the indicated GFP-tagged SNX-BAR proteins precipitating the endogenous CI-MPR from HEK293 cells. (D) GFP-trap IPs of the GFP-tagged WT CI-MPR tail and of the indicated mutant CI-MPR tail constructs were probed for the presence of the indicated endogenous SNXs and endogenous VPS35 by Western blotting. (E) Direct recombinant interaction assay with GST-tagged CI-MPR tail produced in bacteria and the indicated individual His-tagged SNX-BAR proteins produced as secreted proteins in HEK293 cells. (F) Direct recombinant interaction assay with GST-tagged CI-MPR tail produced in bacteria, and the indicated His-tagged SNX-BAR proteins coexpressed as secreted proteins in HEK293 cells. (G) Three CRISPR constructs per indicated gene targeting distinct genomic regions of the respective gene were pooled and cotransfected with a puromycin resistance–encoding plasmid. After puromycin selection and recovery for 3 d, cells were seeded onto coverslips, and CI-MPR localization to the TGN was analyzed by immunofluorescent staining of endogenous CI-MPR (red) and endogenous TGN46 (green). The screen was performed twice, and the colocalization (coloc) between CI-MPR and TGN46 was quantified over 20 images per condition acquired in the two experiments. The efficiency of the respective CRISPR-Cas9 treatment was verified by Western blotting against selected targets. Bars, 10 µm. Error bars indicate SD. *, P

    Techniques Used: Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Mutagenesis, Construct, Recombinant, Produced, CRISPR, Plasmid Preparation, Selection, Staining

    61) Product Images from "Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling"

    Article Title: Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

    Journal: Scientific Reports

    doi: 10.1038/srep13099

    Western blot validation of phosphorylations. ( a ) Western blotting was performed on the cell lysates used for LC-MS/MS analysis. Representative western blots for each antibody are shown. (See Supplementary Fig. 5 for different exposure times). ( b ) Quantitative results of the phosphorylation regulations obtained by western blotting were compared with LC-MS/MS results with different normalizations. Fold-changes (average of triplicates) compared to the control 1 samples are shown. The directions of phosphosite regulations (i.e. up or down) in the CIP2A, RAS, and OA samples (individual replicates) were also compared to the average of control 1 samples. The agreement with western blot was compared between different normalizations using Fisher’s exact test. ( c ) Average correlation coefficients for phosphosites were calculated between the western blotting and LC-MS/MS results on log-transformed data. As the OA samples significantly skewed the data dominating the Pearson’s correlation coefficients, they were excluded from the calculations. Global pairwise normalization led to the highest correlation. ( d ) Spearman’s ρ and Kendall’s τ rank correlation coefficients were also calculated for phosphosites in all samples (i.e. the OA samples included). WB: western blotting, GP: global pairwise, QC: quantile centering, QP: quantile pairwise, GC: global centering, NN: non-normalized, and Ca: casein.
    Figure Legend Snippet: Western blot validation of phosphorylations. ( a ) Western blotting was performed on the cell lysates used for LC-MS/MS analysis. Representative western blots for each antibody are shown. (See Supplementary Fig. 5 for different exposure times). ( b ) Quantitative results of the phosphorylation regulations obtained by western blotting were compared with LC-MS/MS results with different normalizations. Fold-changes (average of triplicates) compared to the control 1 samples are shown. The directions of phosphosite regulations (i.e. up or down) in the CIP2A, RAS, and OA samples (individual replicates) were also compared to the average of control 1 samples. The agreement with western blot was compared between different normalizations using Fisher’s exact test. ( c ) Average correlation coefficients for phosphosites were calculated between the western blotting and LC-MS/MS results on log-transformed data. As the OA samples significantly skewed the data dominating the Pearson’s correlation coefficients, they were excluded from the calculations. Global pairwise normalization led to the highest correlation. ( d ) Spearman’s ρ and Kendall’s τ rank correlation coefficients were also calculated for phosphosites in all samples (i.e. the OA samples included). WB: western blotting, GP: global pairwise, QC: quantile centering, QP: quantile pairwise, GC: global centering, NN: non-normalized, and Ca: casein.

    Techniques Used: Western Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Transformation Assay

    62) Product Images from "Spider genomes provide insight into composition and evolution of venom and silk"

    Article Title: Spider genomes provide insight into composition and evolution of venom and silk

    Journal: Nature Communications

    doi: 10.1038/ncomms4765

    Proteomics. Venn diagrams with number of identified proteins based on 194 LC-MS/MS analyses ( Supplementary Table 3 ). The silk analyses are based on in-solution trypsin digestion, while the body analyses are based on in-gel trypsin digestion. The venom analyses are based on a combination of the two methods. ( a ) Velvet spider. ( b ) Tarantula.
    Figure Legend Snippet: Proteomics. Venn diagrams with number of identified proteins based on 194 LC-MS/MS analyses ( Supplementary Table 3 ). The silk analyses are based on in-solution trypsin digestion, while the body analyses are based on in-gel trypsin digestion. The venom analyses are based on a combination of the two methods. ( a ) Velvet spider. ( b ) Tarantula.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Venomics. ( a ) Coomassie-blue-stained SDS - gel of venom from tarantula and velvet spider, respectively, ( Supplementary Fig. 16 ). The major protein(s) in the bands are: T1, membrane venom metalloendopeptidase-a; T2, putative cysteine-rich venom protease; T3, genicutoxin A1; V1, membrane venom metalloendopeptidase and venom aminopeptidase-a; V2, venom pancreatic-like triacylglycerol lipase-a and c; V3, cysteine-rich venom protease-a; V4, venom phospholipase A2-a; and V5, S.m. Sp2b. In addition to S.m. Sp2b, the V5 band also contains several protoxins. The composition of the visual blue bands was specifically interrogated using the relevant LC-MS/MS analyses of bands from the gels shown in Supplementary Figs 4 and 5 . The data are a sub-fraction of the data shown in Supplementary Data 1 and 2 , where the merged result of LC-MS/MS analyses of gel bands from a complete gel lane is presented. ( b ) The table summarizes quantitative analyses of venom proteins, excluding protoxins (mainly present in the lower bands on the gel in Fig. 3a ). The reason to exclude the protoxins from this analysis is described in the Supplementary Note 2 . The proteases are shown in red, the lipases in grey and the other proteins in shades of blue. Numbers in parentheses refer to the number of variants of the particular protein that were quantified. Individual proteins constituting
    Figure Legend Snippet: Venomics. ( a ) Coomassie-blue-stained SDS - gel of venom from tarantula and velvet spider, respectively, ( Supplementary Fig. 16 ). The major protein(s) in the bands are: T1, membrane venom metalloendopeptidase-a; T2, putative cysteine-rich venom protease; T3, genicutoxin A1; V1, membrane venom metalloendopeptidase and venom aminopeptidase-a; V2, venom pancreatic-like triacylglycerol lipase-a and c; V3, cysteine-rich venom protease-a; V4, venom phospholipase A2-a; and V5, S.m. Sp2b. In addition to S.m. Sp2b, the V5 band also contains several protoxins. The composition of the visual blue bands was specifically interrogated using the relevant LC-MS/MS analyses of bands from the gels shown in Supplementary Figs 4 and 5 . The data are a sub-fraction of the data shown in Supplementary Data 1 and 2 , where the merged result of LC-MS/MS analyses of gel bands from a complete gel lane is presented. ( b ) The table summarizes quantitative analyses of venom proteins, excluding protoxins (mainly present in the lower bands on the gel in Fig. 3a ). The reason to exclude the protoxins from this analysis is described in the Supplementary Note 2 . The proteases are shown in red, the lipases in grey and the other proteins in shades of blue. Numbers in parentheses refer to the number of variants of the particular protein that were quantified. Individual proteins constituting

    Techniques Used: Staining, SDS-Gel, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    63) Product Images from "The PHD and Chromo Domains Regulate the ATPase Activity of the Human Chromatin Remodeler CHD4"

    Article Title: The PHD and Chromo Domains Regulate the ATPase Activity of the Human Chromatin Remodeler CHD4

    Journal: Journal of Molecular Biology

    doi: 10.1016/j.jmb.2012.04.031

    Interdomain cross-links in CHD4. (a) The ATPase domain of CHD4 (AH) was mixed with either the tandem PHD domains (PP) or the tandem PHD plus tandem chromo domains (PP-CC) at a 1:1 molar ratio, and was then incubated with H 12 /D 12 -labeled BS 3 cross-linker in the following protein:cross-linker molar ratios: 1:0 (lane 1), 1:20 (lane 2), 1:50 (lane 3), 1:100 (lane 4), 1:200 (lane 5), and 1:500 (lane 6) for 30 min at room temperature. The protein molecular mass marker (M) is NEB Broad Range. Each reaction was stopped by the addition of 1/10 volume of 1 M Tris, pH 8.0, for 15 min. The reaction mix was separated on a NuPAGE 4–12% Bistris gel and stained with Coomassie Brilliant Blue stain. (b) The above procedure was repeated for constructs PP-CC-AH-D and CC-AH-D separately, each at the following protein:cross-linker ratios: 1:0 (lanes 1 and 6), 1:20 (lanes 2 and 7), 1:50 (lanes 3 and 8), 1:100 (lanes 4 and 9), and 1:200 (lanes 5 and 10). SDS-PAGE bands from lanes 5 and 10 found to have a molecular mass consistent with that of either monomeric PP-CC-AH-D or monomeric CC-AH-D, respectively, are indicated in red boxes. (c) The indicated gel bands were excised, trypsinized, and subjected to LC–MS/MS analysis. Cross-links between the chromo and ATPase-helicase domains within each construct were identified using Xlink-Identifier and are indicated by dotted lines. The sequence of each cross-linked peptide is tabulated below, where each modified lysine is represented in bold, red typeface. The residue numbers of these modified sites are indicated within the table and above the figure. Oxidized methionine residues are denoted in lower case.
    Figure Legend Snippet: Interdomain cross-links in CHD4. (a) The ATPase domain of CHD4 (AH) was mixed with either the tandem PHD domains (PP) or the tandem PHD plus tandem chromo domains (PP-CC) at a 1:1 molar ratio, and was then incubated with H 12 /D 12 -labeled BS 3 cross-linker in the following protein:cross-linker molar ratios: 1:0 (lane 1), 1:20 (lane 2), 1:50 (lane 3), 1:100 (lane 4), 1:200 (lane 5), and 1:500 (lane 6) for 30 min at room temperature. The protein molecular mass marker (M) is NEB Broad Range. Each reaction was stopped by the addition of 1/10 volume of 1 M Tris, pH 8.0, for 15 min. The reaction mix was separated on a NuPAGE 4–12% Bistris gel and stained with Coomassie Brilliant Blue stain. (b) The above procedure was repeated for constructs PP-CC-AH-D and CC-AH-D separately, each at the following protein:cross-linker ratios: 1:0 (lanes 1 and 6), 1:20 (lanes 2 and 7), 1:50 (lanes 3 and 8), 1:100 (lanes 4 and 9), and 1:200 (lanes 5 and 10). SDS-PAGE bands from lanes 5 and 10 found to have a molecular mass consistent with that of either monomeric PP-CC-AH-D or monomeric CC-AH-D, respectively, are indicated in red boxes. (c) The indicated gel bands were excised, trypsinized, and subjected to LC–MS/MS analysis. Cross-links between the chromo and ATPase-helicase domains within each construct were identified using Xlink-Identifier and are indicated by dotted lines. The sequence of each cross-linked peptide is tabulated below, where each modified lysine is represented in bold, red typeface. The residue numbers of these modified sites are indicated within the table and above the figure. Oxidized methionine residues are denoted in lower case.

    Techniques Used: Incubation, Labeling, Marker, Staining, Construct, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Modification

    Limited proteolysis of the PHD, chromo, ATPase-helicase, and DUF1 domains of CHD4. Sites of proteolytic cleavage following LC–MS/MS on construct PP-CC-AH-D are indicated by black arrows with the residue numbers shown above. PHD (P) domains are colored red, chromo (C) domains are colored orange, the ATPase-helicase (AH) domain is colored yellow, and the domains of unknown function (D) are colored green.
    Figure Legend Snippet: Limited proteolysis of the PHD, chromo, ATPase-helicase, and DUF1 domains of CHD4. Sites of proteolytic cleavage following LC–MS/MS on construct PP-CC-AH-D are indicated by black arrows with the residue numbers shown above. PHD (P) domains are colored red, chromo (C) domains are colored orange, the ATPase-helicase (AH) domain is colored yellow, and the domains of unknown function (D) are colored green.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Construct

    64) Product Images from "Extracellular matrix remodelling in response to venous hypertension: proteomics of human varicose veins"

    Article Title: Extracellular matrix remodelling in response to venous hypertension: proteomics of human varicose veins

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvw075

    Characterization of human VSV and results from two different proteomics approaches. ( A ) NSV and VSV were stained with haematoxylin and eosin, with Masson's trichrome and with antibodies for α-smooth muscle actin (SMA). ( B ) ECM proteins of NSV ( n = 6) and VSV ( n = 6) were obtained using our previously published extraction procedure and analysed by LC-MS/MS after separation by gel-LC-MS/MS or in-solution digestion. ( C ) Comparison of differential protein expression in NSV and VSV by gel- and in-solution LC-MS/MS.
    Figure Legend Snippet: Characterization of human VSV and results from two different proteomics approaches. ( A ) NSV and VSV were stained with haematoxylin and eosin, with Masson's trichrome and with antibodies for α-smooth muscle actin (SMA). ( B ) ECM proteins of NSV ( n = 6) and VSV ( n = 6) were obtained using our previously published extraction procedure and analysed by LC-MS/MS after separation by gel-LC-MS/MS or in-solution digestion. ( C ) Comparison of differential protein expression in NSV and VSV by gel- and in-solution LC-MS/MS.

    Techniques Used: Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing

    65) Product Images from "Reaction Pathways in Catechol/Primary Amine Mixtures: A Window on Crosslinking Chemistry"

    Article Title: Reaction Pathways in Catechol/Primary Amine Mixtures: A Window on Crosslinking Chemistry

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0166490

    a) LC-MS chromatograms (monitored at 254 nm) of the reaction mixture of 4MC, PA, and NaIO4 over time. The ratio of 4MC, PA and NaIO4 is 1:3:0.5. b) Extracted ion chromatogram (positive ion mode) corresponding to PMB (m/z = 179.5–180.5) c) ESI-MS spectra [m/z 50–2000] summed over the 10.00–10.53 min retention time window; d) Extracted UV spectra corresponding to the 10.00–10.53 min retention time e) Proposed representative chemical structure of product.
    Figure Legend Snippet: a) LC-MS chromatograms (monitored at 254 nm) of the reaction mixture of 4MC, PA, and NaIO4 over time. The ratio of 4MC, PA and NaIO4 is 1:3:0.5. b) Extracted ion chromatogram (positive ion mode) corresponding to PMB (m/z = 179.5–180.5) c) ESI-MS spectra [m/z 50–2000] summed over the 10.00–10.53 min retention time window; d) Extracted UV spectra corresponding to the 10.00–10.53 min retention time e) Proposed representative chemical structure of product.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    LC-MS analysis for peak identification
    Figure Legend Snippet: LC-MS analysis for peak identification

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    66) Product Images from "Large-scale mass spectrometry-based analysis of Euplotes octocarinatus supports the high frequency of +1 programmed ribosomal frameshift"

    Article Title: Large-scale mass spectrometry-based analysis of Euplotes octocarinatus supports the high frequency of +1 programmed ribosomal frameshift

    Journal: Scientific Reports

    doi: 10.1038/srep33020

    MS analysis of the six frameshift proteins. ( A ) Close-up of the frameshift region is shown. The AAA-UAA motif is shown in bold. The +1 frameshift events are illustrated by curved arrows at the “skipped” nucleotides, which are underlined. The conceptual translations in the 0 reading frame and +1 reading frame are aligned above and below the mRNA sequence, respectively. The amino acids of the peptides identified through mass spectrometry are indicated in red. ( B ) Complete amino acid sequence of the CUFF.27001.1 protein. The peptides identified by MS are indicated in red. The peptide spanning the frameshift site is underlined. The putative frameshift site is highlighted in green. ( C ) LC-MS/MS fragmentation spectrum of the shift site peptide YLMALC KK E from CUFF.27001.1. The insert shows the peptide sequence with ‘b−’ and ‘y−’ type fragment ions that strongly support the shift site peptide identified in the LC-MS/MS analysis. The protein was alkylated with iodoacetamide to protect Cys residues.
    Figure Legend Snippet: MS analysis of the six frameshift proteins. ( A ) Close-up of the frameshift region is shown. The AAA-UAA motif is shown in bold. The +1 frameshift events are illustrated by curved arrows at the “skipped” nucleotides, which are underlined. The conceptual translations in the 0 reading frame and +1 reading frame are aligned above and below the mRNA sequence, respectively. The amino acids of the peptides identified through mass spectrometry are indicated in red. ( B ) Complete amino acid sequence of the CUFF.27001.1 protein. The peptides identified by MS are indicated in red. The peptide spanning the frameshift site is underlined. The putative frameshift site is highlighted in green. ( C ) LC-MS/MS fragmentation spectrum of the shift site peptide YLMALC KK E from CUFF.27001.1. The insert shows the peptide sequence with ‘b−’ and ‘y−’ type fragment ions that strongly support the shift site peptide identified in the LC-MS/MS analysis. The protein was alkylated with iodoacetamide to protect Cys residues.

    Techniques Used: Mass Spectrometry, Sequencing, Liquid Chromatography with Mass Spectroscopy

    MS analysis of the CUFF.27536.1 protein. ( A ) Complete amino acid sequence of the CUFF.27536.1 protein. The peptides identified by MS are indicated in red. The peptide spanning the frameshift site is underlined. The two frameshift sites are highlighted in green. ( B ) LC-MS/MS fragmentation spectrum of the two shift site peptides “VRSKKTGEVRLEKGKQTF” and “IVSMQATKKLLQLQAE” from CUFF.27536.1. The insert shows the peptide sequence with “b−” and “y−” type fragment ions that strongly support the shift site peptides identified in the LC-MS/MS analysis.
    Figure Legend Snippet: MS analysis of the CUFF.27536.1 protein. ( A ) Complete amino acid sequence of the CUFF.27536.1 protein. The peptides identified by MS are indicated in red. The peptide spanning the frameshift site is underlined. The two frameshift sites are highlighted in green. ( B ) LC-MS/MS fragmentation spectrum of the two shift site peptides “VRSKKTGEVRLEKGKQTF” and “IVSMQATKKLLQLQAE” from CUFF.27536.1. The insert shows the peptide sequence with “b−” and “y−” type fragment ions that strongly support the shift site peptides identified in the LC-MS/MS analysis.

    Techniques Used: Mass Spectrometry, Sequencing, Liquid Chromatography with Mass Spectroscopy

    67) Product Images from "Proteome-wide measurement of non-canonical bacterial mistranslation by quantitative mass spectrometry of protein modifications"

    Article Title: Proteome-wide measurement of non-canonical bacterial mistranslation by quantitative mass spectrometry of protein modifications

    Journal: Scientific Reports

    doi: 10.1038/srep28631

    Unbiased protein modification analysis of LC-MS/MS measurements of the WT- and D345A-LeuRS MG1655 strains under microaerobic condition. ( a ) The graph depicts the frequencies of mass-differences (with respect to the unmodified peptide versions) normalized by the total number of spectra (in D345A-LeuRS 462764 and in WT 459446 spectra). The predicted modifications that correspond to the mass differences together with the modified residues are written on top of the graph. The inset shows a 5-fold enlargement of the graph area encompassing the mass difference −28.03 (corresponds to leucine substitution with α‐aminobutyrate). ( b ) Barchart illustrating the localization frequencies of norvaline sites to amino acid residues obtained with the D345A-LeuRS MG1655 strain grown in microaerobic conditions. Sites with localization probability of ≥0.9 are shown.
    Figure Legend Snippet: Unbiased protein modification analysis of LC-MS/MS measurements of the WT- and D345A-LeuRS MG1655 strains under microaerobic condition. ( a ) The graph depicts the frequencies of mass-differences (with respect to the unmodified peptide versions) normalized by the total number of spectra (in D345A-LeuRS 462764 and in WT 459446 spectra). The predicted modifications that correspond to the mass differences together with the modified residues are written on top of the graph. The inset shows a 5-fold enlargement of the graph area encompassing the mass difference −28.03 (corresponds to leucine substitution with α‐aminobutyrate). ( b ) Barchart illustrating the localization frequencies of norvaline sites to amino acid residues obtained with the D345A-LeuRS MG1655 strain grown in microaerobic conditions. Sites with localization probability of ≥0.9 are shown.

    Techniques Used: Modification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    68) Product Images from "Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1"

    Article Title: Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1

    Journal: Journal of the American Society for Mass Spectrometry

    doi: 10.1007/s13361-016-1379-z

    The Q-Exactive plus Orbitrap EMR mass spectrometer produces highly resolved spectra which distinguish between the three DJ-1 variants.
    Figure Legend Snippet: The Q-Exactive plus Orbitrap EMR mass spectrometer produces highly resolved spectra which distinguish between the three DJ-1 variants.

    Techniques Used: Mass Spectrometry

    69) Product Images from "Involvement of Renin-Angiotensin System in Retinopathy of Prematurity - A Possible Target for Therapeutic Intervention"

    Article Title: Involvement of Renin-Angiotensin System in Retinopathy of Prematurity - A Possible Target for Therapeutic Intervention

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0168809

    ERG, OPs Fundus images. Representative graphs for (A) electroretinogram, (B) oscillatory potential. Whereas (C1–6) represents (C1) disease control, (C2) normoxia, (C3) bevacizumab, (C4) lisinopril, (C5) telmisartan group fundus images.
    Figure Legend Snippet: ERG, OPs Fundus images. Representative graphs for (A) electroretinogram, (B) oscillatory potential. Whereas (C1–6) represents (C1) disease control, (C2) normoxia, (C3) bevacizumab, (C4) lisinopril, (C5) telmisartan group fundus images.

    Techniques Used:

    Gene expression analysis. Fig 9A shows the significant multiple folds up regulation of VEGF- vascular endothelial growth factor, HIF1α- Hypoxia inducible factor 1 alpha, angiotensinogen, ACE-Angiotensin Converting Enzyme, AT1 receptor and renin in disease control group in comparison to normal group. Fig 9B, 9C, 9D, 9E, 9F and 9G is representing the normalised expression of HIF 1 α, VEGF, renin, ACE, angiotensinogen and AT1 receptor in various test groups in comparison to disease control. Data is represented as mean ±SEM, significant difference found in comparison with respective control (***p≤0.001, **p≤0.01, *p≤0.05), using Rest (relative expression software tool). DC-disease control, BVZ-bevacizumab, LIS-lisinopril, TELM-telmisartan (n = 9).
    Figure Legend Snippet: Gene expression analysis. Fig 9A shows the significant multiple folds up regulation of VEGF- vascular endothelial growth factor, HIF1α- Hypoxia inducible factor 1 alpha, angiotensinogen, ACE-Angiotensin Converting Enzyme, AT1 receptor and renin in disease control group in comparison to normal group. Fig 9B, 9C, 9D, 9E, 9F and 9G is representing the normalised expression of HIF 1 α, VEGF, renin, ACE, angiotensinogen and AT1 receptor in various test groups in comparison to disease control. Data is represented as mean ±SEM, significant difference found in comparison with respective control (***p≤0.001, **p≤0.01, *p≤0.05), using Rest (relative expression software tool). DC-disease control, BVZ-bevacizumab, LIS-lisinopril, TELM-telmisartan (n = 9).

    Techniques Used: Expressing, Software

    Levels of Lisinopril Telmisartan. Fig 10A shows the telmisartan levels and fig 10B shows the lisinopril levels in the plasma, vitreous and retina. Data is represented as mean ±SEM, significant difference found in comparison with respective control (***p≤0.001, **p≤0.01, *p≤0.05), using unpaired student t-test (n = 9).
    Figure Legend Snippet: Levels of Lisinopril Telmisartan. Fig 10A shows the telmisartan levels and fig 10B shows the lisinopril levels in the plasma, vitreous and retina. Data is represented as mean ±SEM, significant difference found in comparison with respective control (***p≤0.001, **p≤0.01, *p≤0.05), using unpaired student t-test (n = 9).

    Techniques Used:

    70) Product Images from "YcfDRM is a thermophilic oxygen-dependent ribosomal protein uL16 oxygenase"

    Article Title: YcfDRM is a thermophilic oxygen-dependent ribosomal protein uL16 oxygenase

    Journal: Extremophiles

    doi: 10.1007/s00792-018-1016-9

    YcfD RM is a 2-oxoglutarate-dependent oxygenase. a MALDI–MS spectrum of ycfD RM -dependent hydroxylation of R. marinus uL16 RM fragment (KPVTKKPAEVRMGKGKGSVE). A 16 Da mass shift is consistent with a ycfD RM -dependent oxidative modification. b Amino acid analysis reveals (2 S , 3 R )-hydroxylation of R82. Extracted ion chromatograms ( m / z = 345) from LC–MS analysis of: a synthetic (2 S , 3 S )- and (2 S , 3 R )-3-hydroxy-arginine standards, b – d amino acid hydrolysates from ycfD RM -hydroxylated uL16 RM peptide fragment(red trace) overlaid with hydrolysates from a control peptide (black trace, b ), (2 S , 3 R )-hydroxy-arginine standard (blue trace, c ) or (2 S , 3 S )-hydroxy-arginine standard (yellow trace, d ); e – f amino acid hydrolysates from ycfD RM -hydroxylated uL16 RM spiked with either (2 S , 3 R )-hydroxy-arginine ( e ) or (2 S , 3 S )-hydroxy-arginine ( f ) standards. c MS/MS studies on uL16 RM fragment peptide (KPVTKKPAEVRMGKGKGSVE-NH 2 ) incubated with ycfD RM and co-factors/co-substrates (Fe(II), 2OG and ascorbate) revealed hydroxylation at R82. d Co-factor dependence of ycfD RM :ycfD RM (1 μM) was incubated in a reaction mixture from which co-factors and co-substrates (Fe(II), 2OG and ascorbate at 100 μM, 200 μM and 1 mM, respectively), were systematically removed. Apo- and metallated forms of ycfD RM were tested. The reaction was carried out in 50 mM HEPES (pH 7.5) at 65 °C in triplicates. The mean value is shown, with error bars representing standard deviation. e Reaction scheme of ycfD RM -catalysed (2 S , 3 R )-arginine-3-hydroxylation
    Figure Legend Snippet: YcfD RM is a 2-oxoglutarate-dependent oxygenase. a MALDI–MS spectrum of ycfD RM -dependent hydroxylation of R. marinus uL16 RM fragment (KPVTKKPAEVRMGKGKGSVE). A 16 Da mass shift is consistent with a ycfD RM -dependent oxidative modification. b Amino acid analysis reveals (2 S , 3 R )-hydroxylation of R82. Extracted ion chromatograms ( m / z = 345) from LC–MS analysis of: a synthetic (2 S , 3 S )- and (2 S , 3 R )-3-hydroxy-arginine standards, b – d amino acid hydrolysates from ycfD RM -hydroxylated uL16 RM peptide fragment(red trace) overlaid with hydrolysates from a control peptide (black trace, b ), (2 S , 3 R )-hydroxy-arginine standard (blue trace, c ) or (2 S , 3 S )-hydroxy-arginine standard (yellow trace, d ); e – f amino acid hydrolysates from ycfD RM -hydroxylated uL16 RM spiked with either (2 S , 3 R )-hydroxy-arginine ( e ) or (2 S , 3 S )-hydroxy-arginine ( f ) standards. c MS/MS studies on uL16 RM fragment peptide (KPVTKKPAEVRMGKGKGSVE-NH 2 ) incubated with ycfD RM and co-factors/co-substrates (Fe(II), 2OG and ascorbate) revealed hydroxylation at R82. d Co-factor dependence of ycfD RM :ycfD RM (1 μM) was incubated in a reaction mixture from which co-factors and co-substrates (Fe(II), 2OG and ascorbate at 100 μM, 200 μM and 1 mM, respectively), were systematically removed. Apo- and metallated forms of ycfD RM were tested. The reaction was carried out in 50 mM HEPES (pH 7.5) at 65 °C in triplicates. The mean value is shown, with error bars representing standard deviation. e Reaction scheme of ycfD RM -catalysed (2 S , 3 R )-arginine-3-hydroxylation

    Techniques Used: Mass Spectrometry, Modification, Liquid Chromatography with Mass Spectroscopy, Incubation, Standard Deviation

    71) Product Images from "Quantitative analysis of SILAC data sets using spectral counting"

    Article Title: Quantitative analysis of SILAC data sets using spectral counting

    Journal: Proteomics

    doi: 10.1002/pmic.200900684

    Schematic of the SPeCtRA method and overall experimental design. Three separate experiments were conducted in the current study. In experiment 1, proteins from cells grown only in 13 C 6 15 N 4 -Arginine+ 13 C 6 Lysine containing media (Heavy) were digested and analyzed with LC-MS/MS. In experiment 2, proteins from cells grown in heavy and light media were mixed to generate samples with known abundance differences, which were subsequently analyzed with LC-MS/MS. In experiment 3, proteins from cells grown in either heavy normoglycemic media (11mM) or light hyperglycemic media (20mM glucose) were mixed in 1:1 ratio, digested, and analyzed with LC-MS/MS. All LC-MS/MS data were then searched with the SILAC search protocol.
    Figure Legend Snippet: Schematic of the SPeCtRA method and overall experimental design. Three separate experiments were conducted in the current study. In experiment 1, proteins from cells grown only in 13 C 6 15 N 4 -Arginine+ 13 C 6 Lysine containing media (Heavy) were digested and analyzed with LC-MS/MS. In experiment 2, proteins from cells grown in heavy and light media were mixed to generate samples with known abundance differences, which were subsequently analyzed with LC-MS/MS. In experiment 3, proteins from cells grown in either heavy normoglycemic media (11mM) or light hyperglycemic media (20mM glucose) were mixed in 1:1 ratio, digested, and analyzed with LC-MS/MS. All LC-MS/MS data were then searched with the SILAC search protocol.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    72) Product Images from "Aglycon diversity of brain sterylglucosides: structure determination of cholesteryl- and sitosterylglucoside [S]"

    Article Title: Aglycon diversity of brain sterylglucosides: structure determination of cholesteryl- and sitosterylglucoside [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M071480

    Purification of GlcChol from embryonic chicken brain. A: High-performance TLC analysis of the sterylglucoside elution profile from a C18 silica gel column during enrichment, visualized by orcinol staining. GSX, GlcChol-positive fractions, were combined for further purification. B: RPLC-ESI-MS/MS spectra of GSX fraction during neutral-loss scan of [M + NH 4 ] + . Total ion count (TIC) chromatogram, individual MS/MS spectra of indicated peaks. C: Elution profile of purified sterylglucosides from RP-HPLC, as monitored by RPLC-ESI-MS/MS. Monitored precursor-product ion pairs are 566/369, 580/383, and 594/397, depicted with open circles (solid line), filled circles (solid line), and open triangles (dotted line), respectively.
    Figure Legend Snippet: Purification of GlcChol from embryonic chicken brain. A: High-performance TLC analysis of the sterylglucoside elution profile from a C18 silica gel column during enrichment, visualized by orcinol staining. GSX, GlcChol-positive fractions, were combined for further purification. B: RPLC-ESI-MS/MS spectra of GSX fraction during neutral-loss scan of [M + NH 4 ] + . Total ion count (TIC) chromatogram, individual MS/MS spectra of indicated peaks. C: Elution profile of purified sterylglucosides from RP-HPLC, as monitored by RPLC-ESI-MS/MS. Monitored precursor-product ion pairs are 566/369, 580/383, and 594/397, depicted with open circles (solid line), filled circles (solid line), and open triangles (dotted line), respectively.

    Techniques Used: Purification, Thin Layer Chromatography, Staining, Mass Spectrometry, High Performance Liquid Chromatography

    73) Product Images from ""

    Article Title:

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.118.114249

    Identification of proteins by mass spectrometry after ligand-based receptor capture. (A) Schematic view of human proteins identified by LC-MS/MS after LRC experiments with either Gly-probe or Ac-Nle-SP-probe. (B) The amino acid sequence of the human NK 1 receptor is displayed. Peptides that were identified using LC-MS/MS after LRC with Ac-Nle-SP-probe are colored. See Table 1 for further details (1 = blue, 2 = green, 3 = yellow, 4 = purple. 5 = green).
    Figure Legend Snippet: Identification of proteins by mass spectrometry after ligand-based receptor capture. (A) Schematic view of human proteins identified by LC-MS/MS after LRC experiments with either Gly-probe or Ac-Nle-SP-probe. (B) The amino acid sequence of the human NK 1 receptor is displayed. Peptides that were identified using LC-MS/MS after LRC with Ac-Nle-SP-probe are colored. See Table 1 for further details (1 = blue, 2 = green, 3 = yellow, 4 = purple. 5 = green).

    Techniques Used: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Sequencing

    74) Product Images from "Evidence for Endogenous Formation of the Hepatocarcinogen N-Nitrosodihydrouracil in Rats Treated with Dihydrouracil and Sodium Nitrite: a Potential Source of Human Hepatic DNA Carboxyethylation"

    Article Title: Evidence for Endogenous Formation of the Hepatocarcinogen N-Nitrosodihydrouracil in Rats Treated with Dihydrouracil and Sodium Nitrite: a Potential Source of Human Hepatic DNA Carboxyethylation

    Journal: Chemico-biological interactions

    doi: 10.1016/j.cbi.2013.07.010

    Chromatograms obtained upon LC-ESI-MS/MS-SRM analysis of a hydrolysate of rat liver DNA for 7-CEGua, as its methyl ester. SRM was carried out at m/z 238→152 for 7-CEGua methyl ester, shown in the upper panels, and at m/z 243→157 for the internal standard [ 15 N 5 ]7-CEGua methyl ester, shown in the lower panels. ( A ) Chromatograms obtained from the analysis of hepatic DNA from a control animal. ( B ) Chromatograms obtained from the analysis of hepatic DNA from a rat exposed to DHU. ( C ) Chromatograms obtained from the analysis of hepatic DNA from a rat exposed to DHU + NaNO 2.
    Figure Legend Snippet: Chromatograms obtained upon LC-ESI-MS/MS-SRM analysis of a hydrolysate of rat liver DNA for 7-CEGua, as its methyl ester. SRM was carried out at m/z 238→152 for 7-CEGua methyl ester, shown in the upper panels, and at m/z 243→157 for the internal standard [ 15 N 5 ]7-CEGua methyl ester, shown in the lower panels. ( A ) Chromatograms obtained from the analysis of hepatic DNA from a control animal. ( B ) Chromatograms obtained from the analysis of hepatic DNA from a rat exposed to DHU. ( C ) Chromatograms obtained from the analysis of hepatic DNA from a rat exposed to DHU + NaNO 2.

    Techniques Used: Mass Spectrometry

    75) Product Images from "The Phospholipid:Diacylglycerol Acyltransferase Lro1 Is Responsible for Hepatitis C Virus Core-Induced Lipid Droplet Formation in a Yeast Model System"

    Article Title: The Phospholipid:Diacylglycerol Acyltransferase Lro1 Is Responsible for Hepatitis C Virus Core-Induced Lipid Droplet Formation in a Yeast Model System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0159324

    Lipid analysis for the core-expressing yeast cells. (A) TLC analyses of the neutral lipids were performed as described previously [ 27 ]. The positions of ergosterol (ERG), sterol ester (SE) and triacylglycerol (TAG) are indicated. Lipids species found in wild-type cells, dga1Δ cells and lro1Δ cells carrying the pKT10-GAL-core plasmid before (raffinose) and after 3 h of culture with galactose (Galactose) compared by TLC analysis. (B) TAG species found in wild-type (black bars), dga1Δ (green bars) and lro1Δ (red bars) cells carrying the pKT10-GAL-core plasmid before (raffinose) and after 3 h of culture with galactose (Galactose) by LC/MS analysis are depicted, and their abundances are compared. The signal intensity unit for the mass spectrometry detector is indicated as arbitrary unit (AU) for quantification of the abundance of specific m/z of the indicated lipid species.
    Figure Legend Snippet: Lipid analysis for the core-expressing yeast cells. (A) TLC analyses of the neutral lipids were performed as described previously [ 27 ]. The positions of ergosterol (ERG), sterol ester (SE) and triacylglycerol (TAG) are indicated. Lipids species found in wild-type cells, dga1Δ cells and lro1Δ cells carrying the pKT10-GAL-core plasmid before (raffinose) and after 3 h of culture with galactose (Galactose) compared by TLC analysis. (B) TAG species found in wild-type (black bars), dga1Δ (green bars) and lro1Δ (red bars) cells carrying the pKT10-GAL-core plasmid before (raffinose) and after 3 h of culture with galactose (Galactose) by LC/MS analysis are depicted, and their abundances are compared. The signal intensity unit for the mass spectrometry detector is indicated as arbitrary unit (AU) for quantification of the abundance of specific m/z of the indicated lipid species.

    Techniques Used: Expressing, Thin Layer Chromatography, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

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    Quantitation Assay:

    Article Title: Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology
    Article Snippet: Native MS Analysis Identity of aL58ONBY and its decaging efficiency were analyzed by online buffer exchange MS using an UltiMate™ 3000 RSLC (Thermo Fisher ScientificWaltham, MA, USA) coupled to an Exactive Plus EMR Orbitrap instrument (Thermo Fisher ScientificWaltham, MA, USA) modified to incorporate a quadrupole mass filter and allow for surface-induced dissociation [ ]. aL58ONBY was either analyzed in its “as isolated” state or after exposure to UV light (UVP BL-15; Analytik Jena US, Jena, Germany; CA 91786) for 20 min. Next, 100 pmol protein were injected and online buffer was exchanged to 200 mM ammonium acetate, pH 6.8 (AmAc) by a self-packed buffer exchange column [ ] (P6 polyacrylamide gel, BioRad, Hercules, CA, USA) at a flow-rate of 100 µL per min. Mass spectra were recorded for 1000–8000 m/z at 35,000 resolution as defined at 200 m/z . .. Only m/z corresponding to the monomer were considered for deconvolution and subsequent relative quantitation.

    Spectrophotometry:

    Article Title: Design, Synthesis and Evaluation of Oxazaborine Inhibitors of the NLRP3 Inflammasome
    Article Snippet: Accurate mass determination was carried out on a Thermo Exactive Plus EMR Orbitrap LC–MS system. .. Infrared spectroscopy was recorded on a JASCO FT/IR‐4100 spectrophotometer using the Spectra Manager II (JASCO) software package.

    Purification:

    Article Title: Insights into Brain Glycogen Metabolism
    Article Snippet: bGP was purified, and the His tag was removed as described above. .. Native MS was performed on samples containing bGP (1 mg/ml) and phosphorylated bGP (1 mg/ml) using an Exactive Plus EMR mass spectrometer (Thermo Fisher Scientific), which allows analysis of proteins and complexes in native-like states ( ).

    Nuclear Magnetic Resonance:

    Article Title: Design, Synthesis and Evaluation of Oxazaborine Inhibitors of the NLRP3 Inflammasome
    Article Snippet: 19 F NMR chemical shifts were referenced using the deuterium lock signal of the solvent. .. Accurate mass determination was carried out on a Thermo Exactive Plus EMR Orbitrap LC–MS system.

    other:

    Article Title: Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1
    Article Snippet: Stability assays were performed on the Q-Exactive Plus Orbitrap EMR at 17,500 resolution, and the trapping gas pressure was set to 1.

    Article Title: Structural characterization of missense mutations using high resolution mass spectrometry: A case study of the Parkinson’s-related protein, DJ-1
    Article Snippet: The Q-Exactive Plus Orbitrap EMR was operated at capillary voltage 1.7 kV, and argon was used as the collision gas in the higher energy collision-induced dissociation (HCD) cell.

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Design, Synthesis and Evaluation of Oxazaborine Inhibitors of the NLRP3 Inflammasome
    Article Snippet: .. Accurate mass determination was carried out on a Thermo Exactive Plus EMR Orbitrap LC–MS system. ..

    Transmission Assay:

    Article Title: Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology
    Article Snippet: Native MS Analysis Identity of aL58ONBY and its decaging efficiency were analyzed by online buffer exchange MS using an UltiMate™ 3000 RSLC (Thermo Fisher ScientificWaltham, MA, USA) coupled to an Exactive Plus EMR Orbitrap instrument (Thermo Fisher ScientificWaltham, MA, USA) modified to incorporate a quadrupole mass filter and allow for surface-induced dissociation [ ]. aL58ONBY was either analyzed in its “as isolated” state or after exposure to UV light (UVP BL-15; Analytik Jena US, Jena, Germany; CA 91786) for 20 min. Next, 100 pmol protein were injected and online buffer was exchanged to 200 mM ammonium acetate, pH 6.8 (AmAc) by a self-packed buffer exchange column [ ] (P6 polyacrylamide gel, BioRad, Hercules, CA, USA) at a flow-rate of 100 µL per min. Mass spectra were recorded for 1000–8000 m/z at 35,000 resolution as defined at 200 m/z . .. The injection time was set to 200 ms. Voltages applied to the ion optics were optimized to allow for efficient ion transmission while minimizing unintentional ion activa tion.

    Article Title: Comprehensive Proteoform Characterization of Plasma Complement Component C8αβγ by Hybrid Mass Spectrometry Approaches
    Article Snippet: Samples were analyzed on a modified Exactive Plus Orbitrap instrument with extended mass range (EMR) (Thermo Fisher Scientific, Bremen) using a standard m/z range of 500–10,000, as described in detail previously [ ]. .. The voltage offsets on the transport multi-poles and ion lenses were manually tuned to achieve optimal transmission of protein ions at elevated m/z .

    Modification:

    Article Title: Programmable design of orthogonal protein heterodimers
    Article Snippet: .. Mass spectra were subsequently recorded by nanoESI-MS using an Exactive Plus EMR Orbitrap instrument (Thermo Fisher Scientific) modified to incorporate a quadrupole mass filter and allow surface-induced dissociation , , . .. Sample purity and integrity were first analyzed using a self-packed buffer exchange column (P6 polyacrylamide gel, BioRad, Hercules CA), coupled online to an Exactive Plus EMR Orbitrap instrument (Thermo Fisher Scientific) modified to incorporate a quadrupole mass filter and allow surface-induced dissociation.

    Article Title: Programmable design of orthogonal protein heterodimers
    Article Snippet: .. Sample purity and integrity were first analyzed using a self-packed buffer exchange column (P6 polyacrylamide gel, BioRad, Hercules CA), coupled online to an Exactive Plus EMR Orbitrap instrument (Thermo Fisher Scientific) modified to incorporate a quadrupole mass filter and allow surface-induced dissociation. .. For online buffer-exchange, 200 mM ammonium acetate, pH 6.8 (AmAc) was used as a mobile phase.

    Article Title: Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology
    Article Snippet: .. Native MS Analysis Identity of aL58ONBY and its decaging efficiency were analyzed by online buffer exchange MS using an UltiMate™ 3000 RSLC (Thermo Fisher ScientificWaltham, MA, USA) coupled to an Exactive Plus EMR Orbitrap instrument (Thermo Fisher ScientificWaltham, MA, USA) modified to incorporate a quadrupole mass filter and allow for surface-induced dissociation [ ]. aL58ONBY was either analyzed in its “as isolated” state or after exposure to UV light (UVP BL-15; Analytik Jena US, Jena, Germany; CA 91786) for 20 min. Next, 100 pmol protein were injected and online buffer was exchanged to 200 mM ammonium acetate, pH 6.8 (AmAc) by a self-packed buffer exchange column [ ] (P6 polyacrylamide gel, BioRad, Hercules, CA, USA) at a flow-rate of 100 µL per min. Mass spectra were recorded for 1000–8000 m/z at 35,000 resolution as defined at 200 m/z . .. The injection time was set to 200 ms. Voltages applied to the ion optics were optimized to allow for efficient ion transmission while minimizing unintentional ion activa tion.

    Article Title: Comprehensive Proteoform Characterization of Plasma Complement Component C8αβγ by Hybrid Mass Spectrometry Approaches
    Article Snippet: .. Samples were analyzed on a modified Exactive Plus Orbitrap instrument with extended mass range (EMR) (Thermo Fisher Scientific, Bremen) using a standard m/z range of 500–10,000, as described in detail previously [ ]. .. The voltage offsets on the transport multi-poles and ion lenses were manually tuned to achieve optimal transmission of protein ions at elevated m/z .

    Injection:

    Article Title: Stoichiometry of triple-sieve tRNA editing complex ensures fidelity of aminoacyl-tRNA formation
    Article Snippet: Competition studies were performed on an Exactive EMR Orbitrap MS (Thermo Scientific). .. Typical instrument settings were sample spray voltage at 1.0–1.5 kV, in-source dissociation at 60 V, higher energy collisional dissociation (HCD) voltage at 90 V, desolvation temperature at 250°C, source offset voltage at 20 V, S-lens RF level at 150, injection flatapole DC at 8 V, inter flatapole lens at 8, bent flatapole DC at 6 V, transfer multipole DC tuning offset at 0 V, C-trap entrance lens tuning offset at 5 V, high vacuum pressure at 3.5e−9 mbar, and resolving power at 8750 (FWHM at m/z 200).

    Article Title: Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology
    Article Snippet: .. Native MS Analysis Identity of aL58ONBY and its decaging efficiency were analyzed by online buffer exchange MS using an UltiMate™ 3000 RSLC (Thermo Fisher ScientificWaltham, MA, USA) coupled to an Exactive Plus EMR Orbitrap instrument (Thermo Fisher ScientificWaltham, MA, USA) modified to incorporate a quadrupole mass filter and allow for surface-induced dissociation [ ]. aL58ONBY was either analyzed in its “as isolated” state or after exposure to UV light (UVP BL-15; Analytik Jena US, Jena, Germany; CA 91786) for 20 min. Next, 100 pmol protein were injected and online buffer was exchanged to 200 mM ammonium acetate, pH 6.8 (AmAc) by a self-packed buffer exchange column [ ] (P6 polyacrylamide gel, BioRad, Hercules, CA, USA) at a flow-rate of 100 µL per min. Mass spectra were recorded for 1000–8000 m/z at 35,000 resolution as defined at 200 m/z . .. The injection time was set to 200 ms. Voltages applied to the ion optics were optimized to allow for efficient ion transmission while minimizing unintentional ion activa tion.

    Software:

    Article Title: Design, Synthesis and Evaluation of Oxazaborine Inhibitors of the NLRP3 Inflammasome
    Article Snippet: Accurate mass determination was carried out on a Thermo Exactive Plus EMR Orbitrap LC–MS system. .. Infrared spectroscopy was recorded on a JASCO FT/IR‐4100 spectrophotometer using the Spectra Manager II (JASCO) software package.

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    Close your uncertainty gap with HRAM high resolution accurate mass spectrometry Today s forensic toxicology and clinical research laboratory demands more application versatility data accuracy and value from the LC
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    77
    Thermo Fisher lc ms ms analysis all peptide
    GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif <t>analysis</t> for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr <t>peptide</t> abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) <t>MS/MS</t> spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).
    Lc Ms Ms Analysis All Peptide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher lc ms analysis lc ms
    Chromatographic profile of the Impatiens glandulifera Royle methanolic extract of the roots measured as total ion current (TIC) by <t>LC-MS</t> (APCI). MS spectrum of THNG is shown on the Figure 3 .
    Lc Ms Analysis Lc Ms, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher lc esi ms ms
    Hsc70 is a novel neuronal interaction partner of NF-κB. A. Porcine brain extracts were immunoprecipitated with anti NF-κB <t>p65</t> antibody or isotype control on protein G sepharose in presence of cross-linker. The IP were separated in a 1D SDS gel. Each lane (p65 precipitate and control) were cut into 36 slices and prepared for MS by trypsin digestion. All 36 slices were analyzed by MS. Seven samples in range of 95 to 60 and 27 to 24 kDa were additionally analyzed by <t>LC-ESI-MS/MS.</t> The hits identified by MS included the heat shock cognate Hsc70 as a potential interaction partner of NF-κB p65. B. HEK293 co-transfected with p65-flag and Hsc70-myc or IκBε-myc were lysed followed by co-immunoprecipitation in presence of cross-linker using αmyc (IP) antibody with subsequent WB analysis. A clear interaction band (WB: αFlag) was detectable if myc-tagged IκBε and flag-tagged NF-κB p65 were co-transfected. Similarly, co-transfection of p65-flag and Hsc70-myc resulted in a clear interaction band (WB: αFlag), whereas no band was observed in negative controls (no p65-flag, or no IκBε-myc or Hsc70-myc). Lysates were used as input control. C. Neuronal proteins influence the interaction of NF-κB p65 with Hsc70. IP (αmyc) was performed in presence of cross-linker (DSP) and/or brain lysates with subsequent analysis by western blot. Interaction bands (WB: αFlag) were detectable in cross-linked samples for myc-tagged IκBε and flag tagged NF-κB p65 as well as for Hsc70-myc and NF-κB p65-flag. Combination of cross-linker and brain lysates resulted in stronger interaction band (WB: αFlag) for Hsc70-myc and NF-κB p65-flag. Without cross-linker no interaction bands was detectable.
    Lc Esi Ms Ms, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif analysis for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr peptide abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) MS/MS spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).

    Journal: Journal of Proteome Research

    Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

    doi: 10.1021/acs.jproteome.8b00895

    Figure Lengend Snippet: GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif analysis for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr peptide abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) MS/MS spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).

    Article Snippet: LC–MS/MS Analysis All peptide samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer fronted with an EASY-Spray Source, coupled to an Easy-nLC1000 HPLC pump (Thermo Fisher Scientific).

    Techniques: Sequencing, Mass Spectrometry

    Data processing of product ion triggered MS/MS spectra. (A) A schematic of SEQUEST-HT searches of triggered EThcD and HCD spectra using the second Af1521 replicate of IFN-γ-treated THP-1 cells. (B) Number of peptide-spectrum matches (PSMs) of assigned ADPr and unmodified peptides from the triggered spectra. (C–E) Distribution of isolation interference for product ion triggered or DDA PSMs. (F) Number of ADPr peptides with high confidence detected by either EThcD or HCD. (G) Venn diagrams comparing ADPr peptide identifications between EThcD and HCD for all ADPr peptides, and those with > 95% ADPr acceptor site probability.

    Journal: Journal of Proteome Research

    Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

    doi: 10.1021/acs.jproteome.8b00895

    Figure Lengend Snippet: Data processing of product ion triggered MS/MS spectra. (A) A schematic of SEQUEST-HT searches of triggered EThcD and HCD spectra using the second Af1521 replicate of IFN-γ-treated THP-1 cells. (B) Number of peptide-spectrum matches (PSMs) of assigned ADPr and unmodified peptides from the triggered spectra. (C–E) Distribution of isolation interference for product ion triggered or DDA PSMs. (F) Number of ADPr peptides with high confidence detected by either EThcD or HCD. (G) Venn diagrams comparing ADPr peptide identifications between EThcD and HCD for all ADPr peptides, and those with > 95% ADPr acceptor site probability.

    Article Snippet: LC–MS/MS Analysis All peptide samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer fronted with an EASY-Spray Source, coupled to an Easy-nLC1000 HPLC pump (Thermo Fisher Scientific).

    Techniques: Mass Spectrometry, Isolation

    Chromatographic profile of the Impatiens glandulifera Royle methanolic extract of the roots measured as total ion current (TIC) by LC-MS (APCI). MS spectrum of THNG is shown on the Figure 3 .

    Journal: Molecules

    Article Title: Separation and Identification of 1,2,4-Trihydroxynaphthalene-1-O-glucoside in Impatiens glandulifera Royle

    doi: 10.3390/molecules18078429

    Figure Lengend Snippet: Chromatographic profile of the Impatiens glandulifera Royle methanolic extract of the roots measured as total ion current (TIC) by LC-MS (APCI). MS spectrum of THNG is shown on the Figure 3 .

    Article Snippet: LC-MS Analysis LC-MS was performed using LCQ Accela Fleet (Thermo Fisher Scientific, San Jose, CA, USA) with atmospheric pressure chemical (APCI) and a photodiode array detector.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Hsc70 is a novel neuronal interaction partner of NF-κB. A. Porcine brain extracts were immunoprecipitated with anti NF-κB p65 antibody or isotype control on protein G sepharose in presence of cross-linker. The IP were separated in a 1D SDS gel. Each lane (p65 precipitate and control) were cut into 36 slices and prepared for MS by trypsin digestion. All 36 slices were analyzed by MS. Seven samples in range of 95 to 60 and 27 to 24 kDa were additionally analyzed by LC-ESI-MS/MS. The hits identified by MS included the heat shock cognate Hsc70 as a potential interaction partner of NF-κB p65. B. HEK293 co-transfected with p65-flag and Hsc70-myc or IκBε-myc were lysed followed by co-immunoprecipitation in presence of cross-linker using αmyc (IP) antibody with subsequent WB analysis. A clear interaction band (WB: αFlag) was detectable if myc-tagged IκBε and flag-tagged NF-κB p65 were co-transfected. Similarly, co-transfection of p65-flag and Hsc70-myc resulted in a clear interaction band (WB: αFlag), whereas no band was observed in negative controls (no p65-flag, or no IκBε-myc or Hsc70-myc). Lysates were used as input control. C. Neuronal proteins influence the interaction of NF-κB p65 with Hsc70. IP (αmyc) was performed in presence of cross-linker (DSP) and/or brain lysates with subsequent analysis by western blot. Interaction bands (WB: αFlag) were detectable in cross-linked samples for myc-tagged IκBε and flag tagged NF-κB p65 as well as for Hsc70-myc and NF-κB p65-flag. Combination of cross-linker and brain lysates resulted in stronger interaction band (WB: αFlag) for Hsc70-myc and NF-κB p65-flag. Without cross-linker no interaction bands was detectable.

    Journal: PLoS ONE

    Article Title: Hsc70 Is a Novel Interactor of NF-kappaB p65 in Living Hippocampal Neurons

    doi: 10.1371/journal.pone.0065280

    Figure Lengend Snippet: Hsc70 is a novel neuronal interaction partner of NF-κB. A. Porcine brain extracts were immunoprecipitated with anti NF-κB p65 antibody or isotype control on protein G sepharose in presence of cross-linker. The IP were separated in a 1D SDS gel. Each lane (p65 precipitate and control) were cut into 36 slices and prepared for MS by trypsin digestion. All 36 slices were analyzed by MS. Seven samples in range of 95 to 60 and 27 to 24 kDa were additionally analyzed by LC-ESI-MS/MS. The hits identified by MS included the heat shock cognate Hsc70 as a potential interaction partner of NF-κB p65. B. HEK293 co-transfected with p65-flag and Hsc70-myc or IκBε-myc were lysed followed by co-immunoprecipitation in presence of cross-linker using αmyc (IP) antibody with subsequent WB analysis. A clear interaction band (WB: αFlag) was detectable if myc-tagged IκBε and flag-tagged NF-κB p65 were co-transfected. Similarly, co-transfection of p65-flag and Hsc70-myc resulted in a clear interaction band (WB: αFlag), whereas no band was observed in negative controls (no p65-flag, or no IκBε-myc or Hsc70-myc). Lysates were used as input control. C. Neuronal proteins influence the interaction of NF-κB p65 with Hsc70. IP (αmyc) was performed in presence of cross-linker (DSP) and/or brain lysates with subsequent analysis by western blot. Interaction bands (WB: αFlag) were detectable in cross-linked samples for myc-tagged IκBε and flag tagged NF-κB p65 as well as for Hsc70-myc and NF-κB p65-flag. Combination of cross-linker and brain lysates resulted in stronger interaction band (WB: αFlag) for Hsc70-myc and NF-κB p65-flag. Without cross-linker no interaction bands was detectable.

    Article Snippet: MALDI-MS and LC-ESI-MS/MS revealed an interaction of p65 with compounds of the endocytosis network: clathrin and dynamin-1, microtubule subunits or associated proteins like beta 5-tubulin, tubulin alpha 6, beta actin, dihydropyrimidinase-related protein 2, neurofilament and light polypeptide (NEFL) and heat shock proteins HSP90 alpha class A and B (data not shown).

    Techniques: Immunoprecipitation, SDS-Gel, Mass Spectrometry, Transfection, Western Blot, Cotransfection