ltq orbitrap mass spectrometer  (Thermo Fisher)


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    Structured Review

    Thermo Fisher ltq orbitrap mass spectrometer
    Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an <t>LTQ-Orbitrap</t> mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.
    Ltq Orbitrap Mass Spectrometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1665 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65"

    Article Title: PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

    Journal: Open Biology

    doi: 10.1098/rsob.120080

    Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.
    Figure Legend Snippet: Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.

    Techniques Used: Mass Spectrometry, Activation Assay, Expressing, Transfection, Lysis, Immunoprecipitation, SDS Page, Staining, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

    2) Product Images from "Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex"

    Article Title: Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002222

    Quantification of calcium-dependent regulation of phosphorylation sites of Toxoplasma invasion motor complex components. A ) Work flow to identify individual phosphorylation sites and quantitatively assess their responsiveness to calcium signals using a SILAC-based proteomics approach. A 1∶1 mixture of Triton X-100 lysates from “Heavy” (H; Arg4/Lys8)-labeled ethanol-stimulated tachyzoites or
    Figure Legend Snippet: Quantification of calcium-dependent regulation of phosphorylation sites of Toxoplasma invasion motor complex components. A ) Work flow to identify individual phosphorylation sites and quantitatively assess their responsiveness to calcium signals using a SILAC-based proteomics approach. A 1∶1 mixture of Triton X-100 lysates from “Heavy” (H; Arg4/Lys8)-labeled ethanol-stimulated tachyzoites or "Light" (L; Arg0/Lys0)-labeled non-stimulated parasites was generated, and a TiO 2 -enriched phosphopeptide sample of H/L-labeled Toxoplasma invasion motor complexes was prepared and analysed by LC-MS/MS on an LTQ-Orbitrap instrument. Mascot and MaxQuant search engines facilitated subsequent manual identification, phosphosite localization and quantification of proteins or peptides as detailed in materials and mathods. B ) Sypro Ruby-stained SDS-PAGE separation of the relative amounts of light (lane 1) or heavy (lane 2) Triton X-100 whole protein extracts are shown. Intact tachyzoite invasion motor complexes comprising the five major components MyoA, GAP50, GAP45 and MLC1 were precipitated from a 1∶1 H/L mixture by GAP45-specific immuno-affinity chromatography (lane 3).

    Techniques Used: Flow Cytometry, Labeling, Generated, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Staining, SDS Page, Affinity Chromatography

    3) Product Images from "A serotonin-induced N-glycan switch regulates platelet aggregation"

    Article Title: A serotonin-induced N-glycan switch regulates platelet aggregation

    Journal: Scientific Reports

    doi: 10.1038/srep02795

    NSI-MS spectra of permethylated N -glycans from platelets of saline (SAL) (A) and 5-HT-infused (B) mice. An equal number of platelets from WT mice infused for 24 hr with SAL or 5-HT were collected and plasma membrane (PM) was isolated 34 . The N -linked glycans on membrane vesicles were released enzymatically by PNGase F. Released N -glycans were permethylated and profiled by an LTQ Orbitrap Discoverer mass spectrometer equipped with a nanospray ion source. Glycans are detected as singly [M + Na] + , doubly [M + Na] 2+ and triply [M + Na] 3+ charged species. Structural assignments are based on MS/MS fragmentation and known biosynthetic limitation 38 39 . (C) The relative abundance of Neu5Ac on the platelet PM of SAL-infused mice was 2.9-fold higher than Neu5Gc containing glycans, whereas the ratio for platelet PM of 5-HT-infused mice was 3-fold higher in Neu5Gc-containing glycans than Neu5Ac. The total Neu5Ac and Neu5Gc-containing glycans was not different between platelet PM of SAL and 5-HT mice.
    Figure Legend Snippet: NSI-MS spectra of permethylated N -glycans from platelets of saline (SAL) (A) and 5-HT-infused (B) mice. An equal number of platelets from WT mice infused for 24 hr with SAL or 5-HT were collected and plasma membrane (PM) was isolated 34 . The N -linked glycans on membrane vesicles were released enzymatically by PNGase F. Released N -glycans were permethylated and profiled by an LTQ Orbitrap Discoverer mass spectrometer equipped with a nanospray ion source. Glycans are detected as singly [M + Na] + , doubly [M + Na] 2+ and triply [M + Na] 3+ charged species. Structural assignments are based on MS/MS fragmentation and known biosynthetic limitation 38 39 . (C) The relative abundance of Neu5Ac on the platelet PM of SAL-infused mice was 2.9-fold higher than Neu5Gc containing glycans, whereas the ratio for platelet PM of 5-HT-infused mice was 3-fold higher in Neu5Gc-containing glycans than Neu5Ac. The total Neu5Ac and Neu5Gc-containing glycans was not different between platelet PM of SAL and 5-HT mice.

    Techniques Used: Mass Spectrometry, Mouse Assay, Isolation

    4) Product Images from "Yiqihuoxue decoction protects against post-myocardial infarction injury via activation of cardiomyocytes PGC-1α expression"

    Article Title: Yiqihuoxue decoction protects against post-myocardial infarction injury via activation of cardiomyocytes PGC-1α expression

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/s12906-018-2319-1

    The total ion chromatogram of YQHX obtained in negative ionization mode based on UHPLC-LTQ-Orbitrap-MS
    Figure Legend Snippet: The total ion chromatogram of YQHX obtained in negative ionization mode based on UHPLC-LTQ-Orbitrap-MS

    Techniques Used: Mass Spectrometry

    5) Product Images from "The Pneumotoxin 3-Methylindole Is a Substrate and a Mechanism-Based Inactivator of CYP2A13, a Human Cytochrome P450 Enzyme Preferentially Expressed in the Respiratory Tract"

    Article Title: The Pneumotoxin 3-Methylindole Is a Substrate and a Mechanism-Based Inactivator of CYP2A13, a Human Cytochrome P450 Enzyme Preferentially Expressed in the Respiratory Tract

    Journal: Drug Metabolism and Disposition

    doi: 10.1124/dmd.109.027300

    Proposed mass fragmentation patterns for two GSH adducts, GS-A1 and GS-A2, that are formed by CYP2A13-mediated metabolism of 3MI. The mass fragmentation patterns were based on MS n product ion spectra obtained using a LTQ Orbitrap instrument, as described
    Figure Legend Snippet: Proposed mass fragmentation patterns for two GSH adducts, GS-A1 and GS-A2, that are formed by CYP2A13-mediated metabolism of 3MI. The mass fragmentation patterns were based on MS n product ion spectra obtained using a LTQ Orbitrap instrument, as described

    Techniques Used: Mass Spectrometry

    6) Product Images from "Proteomic Analysis Reveals Warburg Effect and Anomalous Metabolism of Glutamine in Pancreatic Cancer Cells"

    Article Title: Proteomic Analysis Reveals Warburg Effect and Anomalous Metabolism of Glutamine in Pancreatic Cancer Cells

    Journal: Journal of proteome research

    doi: 10.1021/pr2009274

    Chromatogram of LC–MS/MS analysis of tryptic peptides from normal pancreatic duct and PANC-1 cells. Twenty microgram tryptic peptides and 100 fmol standard peptide Ang I were loaded to C 18 capillary column, and the eluted peptides were analyzed by LTQ-Orbitrap. For the first iteration of normal duct sample, the retention time of exogenous control Ang I was 78 min, and the retention times of 4 internal control peptides were 26 min, 42 min, 56 min, and 91 min respectively. Labeled peak A is the peptide IWHHTFYNELR (2+ ion, m / z 758.3766) from actin, gamma 1; peak B is the peptide VAPEEHPVLLTEAPLNPK (2+ ion, m / z 977.5364) from actin, gamma 1; peak C is the peptide LGEHNIDVLEGNEQFINAAK (2+ ion, m / z 1106.0545) from trypsin autolysis; peak D is the peptide LCYVALDFEQEMATAASSSSLEK (2+ ion, m / z 1275.5953) from actin, gamma 1. The mass accuracy of these peptides was within 5 ppm. These peptides were also identified in the second and third iterations of normal duct sample, and in 3 iterations of PANC-1 sample with similar retention times (±2.0 min), indicating that the experimental procedure was reproducible and the MS results could be compared.
    Figure Legend Snippet: Chromatogram of LC–MS/MS analysis of tryptic peptides from normal pancreatic duct and PANC-1 cells. Twenty microgram tryptic peptides and 100 fmol standard peptide Ang I were loaded to C 18 capillary column, and the eluted peptides were analyzed by LTQ-Orbitrap. For the first iteration of normal duct sample, the retention time of exogenous control Ang I was 78 min, and the retention times of 4 internal control peptides were 26 min, 42 min, 56 min, and 91 min respectively. Labeled peak A is the peptide IWHHTFYNELR (2+ ion, m / z 758.3766) from actin, gamma 1; peak B is the peptide VAPEEHPVLLTEAPLNPK (2+ ion, m / z 977.5364) from actin, gamma 1; peak C is the peptide LGEHNIDVLEGNEQFINAAK (2+ ion, m / z 1106.0545) from trypsin autolysis; peak D is the peptide LCYVALDFEQEMATAASSSSLEK (2+ ion, m / z 1275.5953) from actin, gamma 1. The mass accuracy of these peptides was within 5 ppm. These peptides were also identified in the second and third iterations of normal duct sample, and in 3 iterations of PANC-1 sample with similar retention times (±2.0 min), indicating that the experimental procedure was reproducible and the MS results could be compared.

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

    7) Product Images from "High resolution tissue imaging on an orbitrap mass spectrometer by desorption electro-spray ionization mass spectrometry (DESI-MS)"

    Article Title: High resolution tissue imaging on an orbitrap mass spectrometer by desorption electro-spray ionization mass spectrometry (DESI-MS)

    Journal: Journal of mass spectrometry : JMS

    doi: 10.1002/jms.1707

    Selected ion images in the negative ion mode of (A) m/z 834.5 on an LTQ ion trap. B) m/z 834.53 and (c) m/z 834.58 collected on an orbitrap
    Figure Legend Snippet: Selected ion images in the negative ion mode of (A) m/z 834.5 on an LTQ ion trap. B) m/z 834.53 and (c) m/z 834.58 collected on an orbitrap

    Techniques Used:

    8) Product Images from "Monte Carlo Simulation-Based Algorithms for Analysis of Shotgun Proteomic Data"

    Article Title: Monte Carlo Simulation-Based Algorithms for Analysis of Shotgun Proteomic Data

    Journal:

    doi: 10.1021/pr800002u

    Distributions of pp2 MCS score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap
    Figure Legend Snippet: Distributions of pp2 MCS score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap

    Techniques Used: Mass Spectrometry

    ROC curves of pp2 MCS and pp tag from the MCS-based score models in MassMatrix along with other statistical scores from Mascot, OMSSA and X!Tandem for (a) the LCQ data set and (b) the LTQ-Orbitrap data set.
    Figure Legend Snippet: ROC curves of pp2 MCS and pp tag from the MCS-based score models in MassMatrix along with other statistical scores from Mascot, OMSSA and X!Tandem for (a) the LCQ data set and (b) the LTQ-Orbitrap data set.

    Techniques Used:

    Search speed in terms of MS/MS spectra per second for MassMatrix, Mascot, OMSSA, and X!Tandem on a PC with an Intel quad core CPU (2.4 GHz) and Linux operating system. The two merged data sets from LCQ and LTQ-Orbitrap mass spectrometers were searched
    Figure Legend Snippet: Search speed in terms of MS/MS spectra per second for MassMatrix, Mascot, OMSSA, and X!Tandem on a PC with an Intel quad core CPU (2.4 GHz) and Linux operating system. The two merged data sets from LCQ and LTQ-Orbitrap mass spectrometers were searched

    Techniques Used: Mass Spectrometry

    ROC curves of different score standards in MassMatrix for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap mass spectrometer.
    Figure Legend Snippet: ROC curves of different score standards in MassMatrix for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap mass spectrometer.

    Techniques Used: Mass Spectrometry

    Distributions of pp tag score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap
    Figure Legend Snippet: Distributions of pp tag score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap

    Techniques Used: Mass Spectrometry

    9) Product Images from "Monte Carlo Simulation-Based Algorithms for Analysis of Shotgun Proteomic Data"

    Article Title: Monte Carlo Simulation-Based Algorithms for Analysis of Shotgun Proteomic Data

    Journal:

    doi: 10.1021/pr800002u

    Distributions of pp2 MCS score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap
    Figure Legend Snippet: Distributions of pp2 MCS score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap

    Techniques Used: Mass Spectrometry

    ROC curves of pp2 MCS and pp tag from the MCS-based score models in MassMatrix along with other statistical scores from Mascot, OMSSA and X!Tandem for (a) the LCQ data set and (b) the LTQ-Orbitrap data set.
    Figure Legend Snippet: ROC curves of pp2 MCS and pp tag from the MCS-based score models in MassMatrix along with other statistical scores from Mascot, OMSSA and X!Tandem for (a) the LCQ data set and (b) the LTQ-Orbitrap data set.

    Techniques Used:

    Search speed in terms of MS/MS spectra per second for MassMatrix, Mascot, OMSSA, and X!Tandem on a PC with an Intel quad core CPU (2.4 GHz) and Linux operating system. The two merged data sets from LCQ and LTQ-Orbitrap mass spectrometers were searched
    Figure Legend Snippet: Search speed in terms of MS/MS spectra per second for MassMatrix, Mascot, OMSSA, and X!Tandem on a PC with an Intel quad core CPU (2.4 GHz) and Linux operating system. The two merged data sets from LCQ and LTQ-Orbitrap mass spectrometers were searched

    Techniques Used: Mass Spectrometry

    ROC curves of different score standards in MassMatrix for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap mass spectrometer.
    Figure Legend Snippet: ROC curves of different score standards in MassMatrix for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap mass spectrometer.

    Techniques Used: Mass Spectrometry

    Distributions of pp tag score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap
    Figure Legend Snippet: Distributions of pp tag score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap

    Techniques Used: Mass Spectrometry

    10) Product Images from "Intestinal epithelial N-acylphosphatidylethanolamine phospholipase D links dietary fat to metabolic adaptations in obesity and steatosis"

    Article Title: Intestinal epithelial N-acylphosphatidylethanolamine phospholipase D links dietary fat to metabolic adaptations in obesity and steatosis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08051-7

    Validation of IEC Napepld deletion. a Napepld mRNA expression in the jejunum, colon, liver and epididymal adipose tissue (EAT) in ND-fed WT and Napepld ∆IEC mice ( n = 8–10). b NAPE-PLD protein levels in the colon. Representative western-blot of NAPE-PLD and β-Actin ( n = 9–10). c Levels of N -acylethanolamines and 2-acylglycerols in small intestinal epithelial cells of ND-fed WT and Napepld ∆IEC mice ( n = 7–9) were determined by using high-performance liquid chromatography-MS using an LTQ Orbitrap mass spectrometer as described in the methods. Dark blue: WT ND mice, light blue: Napepld ∆IEC ND mice. Data are presented as the mean ± s.e.m. *, ** and *** indicate a significant difference versus WT ND (Respectively P
    Figure Legend Snippet: Validation of IEC Napepld deletion. a Napepld mRNA expression in the jejunum, colon, liver and epididymal adipose tissue (EAT) in ND-fed WT and Napepld ∆IEC mice ( n = 8–10). b NAPE-PLD protein levels in the colon. Representative western-blot of NAPE-PLD and β-Actin ( n = 9–10). c Levels of N -acylethanolamines and 2-acylglycerols in small intestinal epithelial cells of ND-fed WT and Napepld ∆IEC mice ( n = 7–9) were determined by using high-performance liquid chromatography-MS using an LTQ Orbitrap mass spectrometer as described in the methods. Dark blue: WT ND mice, light blue: Napepld ∆IEC ND mice. Data are presented as the mean ± s.e.m. *, ** and *** indicate a significant difference versus WT ND (Respectively P

    Techniques Used: Expressing, Mouse Assay, Western Blot, High Performance Liquid Chromatography, Mass Spectrometry

    11) Product Images from "Shotgun Lipidomics Identifies a Paired Rule for the Presence of Isomeric Ether Phospholipid Molecular Species"

    Article Title: Shotgun Lipidomics Identifies a Paired Rule for the Presence of Isomeric Ether Phospholipid Molecular Species

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001368

    Representative negative-ion ESI/MS analyses of bovine heart ethanolamine glycerophospholipid molecular species. Bovine heart lipids were extracted by a modified Bligh and Dyer procedure [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23] . Analyses of PtdEtn molecular species were performed in the negative-ion mode by using an LTQ-Orbitrap mass spectrometer equipped with a Nanomate device as described under “ MATERIALS AND METHODS ”.
    Figure Legend Snippet: Representative negative-ion ESI/MS analyses of bovine heart ethanolamine glycerophospholipid molecular species. Bovine heart lipids were extracted by a modified Bligh and Dyer procedure [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23] . Analyses of PtdEtn molecular species were performed in the negative-ion mode by using an LTQ-Orbitrap mass spectrometer equipped with a Nanomate device as described under “ MATERIALS AND METHODS ”.

    Techniques Used: Mass Spectrometry, Modification, High Performance Liquid Chromatography

    Product ion analyses of synthetic 18∶0-20∶4 plasmenylethanolamine molecular species in the negative-ion mode. Product ion ESI/MS analysis of deprotonated 18∶0-20∶4 plasmenylethanolamine at m/z 750.54 was performed on an LTQ-Orbitrap mass spectrometer with a C-trap using an ion selective window of 1 Th by LTQ. Collision activation in C-trap was carried out with normalized collision energy of 55% and gas pressure of 1 mT. The resultant fragment ions were analyzed in the Orbitrap. The arrow indicates the absence of the 18:0 FA carboxylate in the spectrum after amplifying the position greater than1,000 fold.
    Figure Legend Snippet: Product ion analyses of synthetic 18∶0-20∶4 plasmenylethanolamine molecular species in the negative-ion mode. Product ion ESI/MS analysis of deprotonated 18∶0-20∶4 plasmenylethanolamine at m/z 750.54 was performed on an LTQ-Orbitrap mass spectrometer with a C-trap using an ion selective window of 1 Th by LTQ. Collision activation in C-trap was carried out with normalized collision energy of 55% and gas pressure of 1 mT. The resultant fragment ions were analyzed in the Orbitrap. The arrow indicates the absence of the 18:0 FA carboxylate in the spectrum after amplifying the position greater than1,000 fold.

    Techniques Used: Mass Spectrometry, Activation Assay

    Product ion analyses of individual molecular species present in isolated bovine heart ethanolamine glycerophospholipids. Product ion ESI/MS analyses of PtdEtn molecular species in isolated bovine heart ethanolamine glycerophospholipids at m/z 772.5 (Panel A) and 774.5 (Panel B) were performed by using an LTQ-Orbitrap mass spectrometer. Analyses were conducted using a peak width setting of 1 Th by selection of the molecular ion in the linear ion trap (LTQ), collision activation in the C-trap with a normalized collision energy of 55% and gas pressure of 1 mTorr, and analysis of the resultant product ions in the Orbitrap.
    Figure Legend Snippet: Product ion analyses of individual molecular species present in isolated bovine heart ethanolamine glycerophospholipids. Product ion ESI/MS analyses of PtdEtn molecular species in isolated bovine heart ethanolamine glycerophospholipids at m/z 772.5 (Panel A) and 774.5 (Panel B) were performed by using an LTQ-Orbitrap mass spectrometer. Analyses were conducted using a peak width setting of 1 Th by selection of the molecular ion in the linear ion trap (LTQ), collision activation in the C-trap with a normalized collision energy of 55% and gas pressure of 1 mTorr, and analysis of the resultant product ions in the Orbitrap.

    Techniques Used: Isolation, Mass Spectrometry, Selection, Activation Assay

    12) Product Images from "Shotgun Lipidomics Identifies a Paired Rule for the Presence of Isomeric Ether Phospholipid Molecular Species"

    Article Title: Shotgun Lipidomics Identifies a Paired Rule for the Presence of Isomeric Ether Phospholipid Molecular Species

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001368

    Representative negative-ion ESI/MS analyses of bovine heart ethanolamine glycerophospholipid molecular species. Bovine heart lipids were extracted by a modified Bligh and Dyer procedure [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23] . Analyses of PtdEtn molecular species were performed in the negative-ion mode by using an LTQ-Orbitrap mass spectrometer equipped with a Nanomate device as described under “ MATERIALS AND METHODS ”.
    Figure Legend Snippet: Representative negative-ion ESI/MS analyses of bovine heart ethanolamine glycerophospholipid molecular species. Bovine heart lipids were extracted by a modified Bligh and Dyer procedure [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23] . Analyses of PtdEtn molecular species were performed in the negative-ion mode by using an LTQ-Orbitrap mass spectrometer equipped with a Nanomate device as described under “ MATERIALS AND METHODS ”.

    Techniques Used: Mass Spectrometry, Modification, High Performance Liquid Chromatography

    Product ion analyses of synthetic 18∶0-20∶4 plasmenylethanolamine molecular species in the negative-ion mode. Product ion ESI/MS analysis of deprotonated 18∶0-20∶4 plasmenylethanolamine at m/z 750.54 was performed on an LTQ-Orbitrap mass spectrometer with a C-trap using an ion selective window of 1 Th by LTQ. Collision activation in C-trap was carried out with normalized collision energy of 55% and gas pressure of 1 mT. The resultant fragment ions were analyzed in the Orbitrap. The arrow indicates the absence of the 18:0 FA carboxylate in the spectrum after amplifying the position greater than1,000 fold.
    Figure Legend Snippet: Product ion analyses of synthetic 18∶0-20∶4 plasmenylethanolamine molecular species in the negative-ion mode. Product ion ESI/MS analysis of deprotonated 18∶0-20∶4 plasmenylethanolamine at m/z 750.54 was performed on an LTQ-Orbitrap mass spectrometer with a C-trap using an ion selective window of 1 Th by LTQ. Collision activation in C-trap was carried out with normalized collision energy of 55% and gas pressure of 1 mT. The resultant fragment ions were analyzed in the Orbitrap. The arrow indicates the absence of the 18:0 FA carboxylate in the spectrum after amplifying the position greater than1,000 fold.

    Techniques Used: Mass Spectrometry, Activation Assay

    Product ion analyses of individual molecular species present in isolated bovine heart ethanolamine glycerophospholipids. Product ion ESI/MS analyses of PtdEtn molecular species in isolated bovine heart ethanolamine glycerophospholipids at m/z 772.5 (Panel A) and 774.5 (Panel B) were performed by using an LTQ-Orbitrap mass spectrometer. Analyses were conducted using a peak width setting of 1 Th by selection of the molecular ion in the linear ion trap (LTQ), collision activation in the C-trap with a normalized collision energy of 55% and gas pressure of 1 mTorr, and analysis of the resultant product ions in the Orbitrap.
    Figure Legend Snippet: Product ion analyses of individual molecular species present in isolated bovine heart ethanolamine glycerophospholipids. Product ion ESI/MS analyses of PtdEtn molecular species in isolated bovine heart ethanolamine glycerophospholipids at m/z 772.5 (Panel A) and 774.5 (Panel B) were performed by using an LTQ-Orbitrap mass spectrometer. Analyses were conducted using a peak width setting of 1 Th by selection of the molecular ion in the linear ion trap (LTQ), collision activation in the C-trap with a normalized collision energy of 55% and gas pressure of 1 mTorr, and analysis of the resultant product ions in the Orbitrap.

    Techniques Used: Isolation, Mass Spectrometry, Selection, Activation Assay

    13) Product Images from "Yiqihuoxue decoction protects against post-myocardial infarction injury via activation of cardiomyocytes PGC-1α expression"

    Article Title: Yiqihuoxue decoction protects against post-myocardial infarction injury via activation of cardiomyocytes PGC-1α expression

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/s12906-018-2319-1

    The total ion chromatogram of YQHX obtained in negative ionization mode based on UHPLC-LTQ-Orbitrap-MS
    Figure Legend Snippet: The total ion chromatogram of YQHX obtained in negative ionization mode based on UHPLC-LTQ-Orbitrap-MS

    Techniques Used: Mass Spectrometry

    14) Product Images from "Kinetic Study of Neuropeptide Y (NPY) Proteolysis in Blood and Identification of NPY3–35"

    Article Title: Kinetic Study of Neuropeptide Y (NPY) Proteolysis in Blood and Identification of NPY3–35

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.035253

    Mass spectrometry characterization of NPY 3–35 . A , LTQ Orbitrap, full-MS spectra m / z 350–2000; signals of NPY 3–35 , NPY 3–36 , and NPY 2–36 (charge states +3, +4, and +5 are in bold ). B , LC-MS chromatograms for the most
    Figure Legend Snippet: Mass spectrometry characterization of NPY 3–35 . A , LTQ Orbitrap, full-MS spectra m / z 350–2000; signals of NPY 3–35 , NPY 3–36 , and NPY 2–36 (charge states +3, +4, and +5 are in bold ). B , LC-MS chromatograms for the most

    Techniques Used: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    15) Product Images from "Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors"

    Article Title: Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors

    Journal: Genome Biology

    doi: 10.1186/gb-2006-7-8-r72

    Data comparison between LTQ-FT and LTQ-Orbitrap spectrometry. The numbers of peptides for the top six identified proteins (LTQ-FT data) were compared between the two methods. Except for the protein Apolipoprotein B100, we observed a significant increase in the number of peptides identified with the LTQ-Orbitrap. This pattern was observed for most of the proteins identified with more then three peptides in the LTQ-FT. Light gray bars represent LTQ-FT data, dark gray represents LTQ-Orbitrap data.
    Figure Legend Snippet: Data comparison between LTQ-FT and LTQ-Orbitrap spectrometry. The numbers of peptides for the top six identified proteins (LTQ-FT data) were compared between the two methods. Except for the protein Apolipoprotein B100, we observed a significant increase in the number of peptides identified with the LTQ-Orbitrap. This pattern was observed for most of the proteins identified with more then three peptides in the LTQ-FT. Light gray bars represent LTQ-FT data, dark gray represents LTQ-Orbitrap data.

    Techniques Used:

    Approach used for tear fluid analysis. The tear fluid was analyzed by both in-solution digestion (1 and 4 μl) and one-dimensional gel separation combined with MS (GeLC-MS; 2 lanes of 4 μl each) on a LTQ-FT, and also through GeLC-MS on a LTQ-Orbitrap. The numbers indicate the bands according to the slicing pattern used for sample fractionation prior to in situ digestion.
    Figure Legend Snippet: Approach used for tear fluid analysis. The tear fluid was analyzed by both in-solution digestion (1 and 4 μl) and one-dimensional gel separation combined with MS (GeLC-MS; 2 lanes of 4 μl each) on a LTQ-FT, and also through GeLC-MS on a LTQ-Orbitrap. The numbers indicate the bands according to the slicing pattern used for sample fractionation prior to in situ digestion.

    Techniques Used: Mass Spectrometry, Fractionation, In Situ

    16) Product Images from "Shotgun Lipidomics Identifies a Paired Rule for the Presence of Isomeric Ether Phospholipid Molecular Species"

    Article Title: Shotgun Lipidomics Identifies a Paired Rule for the Presence of Isomeric Ether Phospholipid Molecular Species

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001368

    Representative negative-ion ESI/MS analyses of bovine heart ethanolamine glycerophospholipid molecular species. Bovine heart lipids were extracted by a modified Bligh and Dyer procedure [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23] . Analyses of PtdEtn molecular species were performed in the negative-ion mode by using an LTQ-Orbitrap mass spectrometer equipped with a Nanomate device as described under “ MATERIALS AND METHODS ”.
    Figure Legend Snippet: Representative negative-ion ESI/MS analyses of bovine heart ethanolamine glycerophospholipid molecular species. Bovine heart lipids were extracted by a modified Bligh and Dyer procedure [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23] . Analyses of PtdEtn molecular species were performed in the negative-ion mode by using an LTQ-Orbitrap mass spectrometer equipped with a Nanomate device as described under “ MATERIALS AND METHODS ”.

    Techniques Used: Mass Spectrometry, Modification, High Performance Liquid Chromatography

    Product ion analyses of synthetic 18∶0-20∶4 plasmenylethanolamine molecular species in the negative-ion mode. Product ion ESI/MS analysis of deprotonated 18∶0-20∶4 plasmenylethanolamine at m/z 750.54 was performed on an LTQ-Orbitrap mass spectrometer with a C-trap using an ion selective window of 1 Th by LTQ. Collision activation in C-trap was carried out with normalized collision energy of 55% and gas pressure of 1 mT. The resultant fragment ions were analyzed in the Orbitrap. The arrow indicates the absence of the 18:0 FA carboxylate in the spectrum after amplifying the position greater than1,000 fold.
    Figure Legend Snippet: Product ion analyses of synthetic 18∶0-20∶4 plasmenylethanolamine molecular species in the negative-ion mode. Product ion ESI/MS analysis of deprotonated 18∶0-20∶4 plasmenylethanolamine at m/z 750.54 was performed on an LTQ-Orbitrap mass spectrometer with a C-trap using an ion selective window of 1 Th by LTQ. Collision activation in C-trap was carried out with normalized collision energy of 55% and gas pressure of 1 mT. The resultant fragment ions were analyzed in the Orbitrap. The arrow indicates the absence of the 18:0 FA carboxylate in the spectrum after amplifying the position greater than1,000 fold.

    Techniques Used: Mass Spectrometry, Activation Assay

    Product ion analyses of individual molecular species present in isolated bovine heart ethanolamine glycerophospholipids. Product ion ESI/MS analyses of PtdEtn molecular species in isolated bovine heart ethanolamine glycerophospholipids at m/z 772.5 (Panel A) and 774.5 (Panel B) were performed by using an LTQ-Orbitrap mass spectrometer. Analyses were conducted using a peak width setting of 1 Th by selection of the molecular ion in the linear ion trap (LTQ), collision activation in the C-trap with a normalized collision energy of 55% and gas pressure of 1 mTorr, and analysis of the resultant product ions in the Orbitrap.
    Figure Legend Snippet: Product ion analyses of individual molecular species present in isolated bovine heart ethanolamine glycerophospholipids. Product ion ESI/MS analyses of PtdEtn molecular species in isolated bovine heart ethanolamine glycerophospholipids at m/z 772.5 (Panel A) and 774.5 (Panel B) were performed by using an LTQ-Orbitrap mass spectrometer. Analyses were conducted using a peak width setting of 1 Th by selection of the molecular ion in the linear ion trap (LTQ), collision activation in the C-trap with a normalized collision energy of 55% and gas pressure of 1 mTorr, and analysis of the resultant product ions in the Orbitrap.

    Techniques Used: Isolation, Mass Spectrometry, Selection, Activation Assay

    17) Product Images from "Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags"

    Article Title: Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags

    Journal: Analytical chemistry

    doi: 10.1021/ac702328x

    The UStags process for identifying unique peptide sequences from precise LC-MS and MS/MS experiments. Yeast whole cell tryptic digest was analyzed using an LC-LTQ-Orbitrap platform; resulting LC-MS/MS dataset was searched using SEQUEST against yeast sequence database. Amino acid residue sequencing (see Supplementary Methods) was applied to obtain accurate AA residue assignments and sequences. These initial unique sequences were examined using the residue replacement filter (RRF; see Supplementary Methods), illustrated here for TSGTLEMNLK(1). This sequence was found in a single protein (the number in the parenthesis); however, an isobaric variant sequence SSGTLEMNLK potentially exists in another yeast protein when modifications are considered (i.e., Ser-me-ester has the same mass as Thr). Due to the ambiguity this sequence cannot serve as an UStag (i.e., only sequences remaining after the RRF are considered UStags).
    Figure Legend Snippet: The UStags process for identifying unique peptide sequences from precise LC-MS and MS/MS experiments. Yeast whole cell tryptic digest was analyzed using an LC-LTQ-Orbitrap platform; resulting LC-MS/MS dataset was searched using SEQUEST against yeast sequence database. Amino acid residue sequencing (see Supplementary Methods) was applied to obtain accurate AA residue assignments and sequences. These initial unique sequences were examined using the residue replacement filter (RRF; see Supplementary Methods), illustrated here for TSGTLEMNLK(1). This sequence was found in a single protein (the number in the parenthesis); however, an isobaric variant sequence SSGTLEMNLK potentially exists in another yeast protein when modifications are considered (i.e., Ser-me-ester has the same mass as Thr). Due to the ambiguity this sequence cannot serve as an UStag (i.e., only sequences remaining after the RRF are considered UStags).

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

    18) Product Images from "Analysis of Chemical Constituents in Wuzi-Yanzong-Wan by UPLC-ESI-LTQ-Orbitrap-MS"

    Article Title: Analysis of Chemical Constituents in Wuzi-Yanzong-Wan by UPLC-ESI-LTQ-Orbitrap-MS

    Journal: Molecules

    doi: 10.3390/molecules201219765

    Flavonoids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.
    Figure Legend Snippet: Flavonoids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.

    Techniques Used: Mass Spectrometry

    Organic acids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.
    Figure Legend Snippet: Organic acids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.

    Techniques Used: Mass Spectrometry

    Total ion chromatogram (TIC) of WZYZW in negative ion mode ( a ) and positive ion mode ( b ) using UPLC-ESI-LTQ-Orbitrap-MS.
    Figure Legend Snippet: Total ion chromatogram (TIC) of WZYZW in negative ion mode ( a ) and positive ion mode ( b ) using UPLC-ESI-LTQ-Orbitrap-MS.

    Techniques Used: Mass Spectrometry

    Phenylpropanoids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.
    Figure Legend Snippet: Phenylpropanoids identified in WZYZW by UPLC-ESI-LTQ-Orbitrap-MS.

    Techniques Used: Mass Spectrometry

    19) Product Images from "Phytochemical Composition, Hepatoprotective, and Antioxidant Activities of Phyllodium pulchellum (L.) Desv"

    Article Title: Phytochemical Composition, Hepatoprotective, and Antioxidant Activities of Phyllodium pulchellum (L.) Desv

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23061361

    The total ion chromatograms (TIC) of the total flavonoids of P. pulchellum by high-performance liquid chromatography-linear ion trap quadrupole-Orbitrap-mass spectrometry (HPLC-LTQ-Orbitrap-MS) in negative ion mode ( A ) and positive ion mode ( B ). The compounds that are confirmed with the isolated reference compounds are marked in red.
    Figure Legend Snippet: The total ion chromatograms (TIC) of the total flavonoids of P. pulchellum by high-performance liquid chromatography-linear ion trap quadrupole-Orbitrap-mass spectrometry (HPLC-LTQ-Orbitrap-MS) in negative ion mode ( A ) and positive ion mode ( B ). The compounds that are confirmed with the isolated reference compounds are marked in red.

    Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry, Isolation

    20) Product Images from "Facile Identification and Quantitation of Protein Phosphorylation via ?-Elimination and Michael Addition with Natural Abundance and Stable Isotope Labeled Thiocholine"

    Article Title: Facile Identification and Quantitation of Protein Phosphorylation via ?-Elimination and Michael Addition with Natural Abundance and Stable Isotope Labeled Thiocholine

    Journal: Analytical chemistry

    doi: 10.1021/ac9015193

    Comparison of the ionization efficiency of the phosphopeptide FQpSEEQQQTEDELQDK (▲) and its thiocholine modified derivative FQS*EEQQQTEDELQDK (■) ”. “pS” indicates the phosphorylation site and “S*” indicates the thiocholine modified site. A . Separation of the peptide mixture using a reverse-phase C 18 column and analysis employing an ESI-LTQ-Orbitrap as described in “Methods”. The full-mass scan extract ion chromatogram (XIC) of ions at m/z 694.983 (+3) and 1031.417 (+2) with normalized ion intensities is presented. The ion at m/z 694.983 corresponds to the triply charged molecular ion (dominant charge state) of FQS*EEQQQTEDELQDK (■) and the ion at m/z 1031.417 corresponds to the doubly charged molecular ion (dominant charge state) of FQpSEEQQQTEDELQDK (▲). The XIC shows a 100-fold increase in ionization efficiency of the peptide after thiocholine modification. B . The full-mass spectrum of the 1:1 peptide mixture obtained with a 4800 MALDI-TOF/TOF Analyzer with normalized ion intensity as described in “Methods”. The ion peak at m/z 2061.52 corresponds to the singly charged molecular ion of FQpSEEQQQTEDELQDK (▲) and the ion peak at m/z 2082.62 corresponds to the singly charged molecular ion of FQS*EEQQQTEDELQDK (■). A 3-fold increase in ionization efficiency of the peptide after thiocholine modification was observed with MALDI-MS.
    Figure Legend Snippet: Comparison of the ionization efficiency of the phosphopeptide FQpSEEQQQTEDELQDK (▲) and its thiocholine modified derivative FQS*EEQQQTEDELQDK (■) ”. “pS” indicates the phosphorylation site and “S*” indicates the thiocholine modified site. A . Separation of the peptide mixture using a reverse-phase C 18 column and analysis employing an ESI-LTQ-Orbitrap as described in “Methods”. The full-mass scan extract ion chromatogram (XIC) of ions at m/z 694.983 (+3) and 1031.417 (+2) with normalized ion intensities is presented. The ion at m/z 694.983 corresponds to the triply charged molecular ion (dominant charge state) of FQS*EEQQQTEDELQDK (■) and the ion at m/z 1031.417 corresponds to the doubly charged molecular ion (dominant charge state) of FQpSEEQQQTEDELQDK (▲). The XIC shows a 100-fold increase in ionization efficiency of the peptide after thiocholine modification. B . The full-mass spectrum of the 1:1 peptide mixture obtained with a 4800 MALDI-TOF/TOF Analyzer with normalized ion intensity as described in “Methods”. The ion peak at m/z 2061.52 corresponds to the singly charged molecular ion of FQpSEEQQQTEDELQDK (▲) and the ion peak at m/z 2082.62 corresponds to the singly charged molecular ion of FQS*EEQQQTEDELQDK (■). A 3-fold increase in ionization efficiency of the peptide after thiocholine modification was observed with MALDI-MS.

    Techniques Used: Modification, Mass Spectrometry

    Fragmentation of the thiocholine modified peptide FQS*EEQQQTEDELQDK A . The MS 2 spectrum of the triply charged molecular ion of FQS*EEQQQTEDELQDK at m/z 694.983 was obtained with an ESI-LTQ-Orbitrap as described in “Methods”. The fragment ion resulting from the neutral loss of trimethylamine from the parent ion was not detected. Shown in the expanded spectrum is an example of the diagnostic triad consisting of the b 5 + ion resulting from peptide bond cleavage ( m/z =722.3) and further neutral loss of trimethylamine (−59Da, m/z =663.2) or the thiocholine thiolate (−119Da, m/z =603.2). “S*” indicates the thiocholine modified site. B . The MS 3 spectrum of the y 14 +2 ion at m/z 904.4 resulting from the fragmentation of the triply charged molecular ion of FQS*EEQQQTEDELQDK at m/z 694.983. The ion peak at m/z 875.1 corresponds to the doubly charged fragment ion generated from the neutral loss of trimethylamine from the parent ion y 14 +2 . C . The MS 2 spectrum of the singly charged molecular ion of FQS*EEQQQTEDELQDK at m/z 2082.62, obtained with a 4800 MALDI-TOF/TOF Analyzer as described in “Methods”. The ion peak at m/z 2023.72 corresponds to the fragment ion resulting from the neutral loss of trimethylamine from the parent ion. Shown in the expanded spectrum are the low-abundance y ions.
    Figure Legend Snippet: Fragmentation of the thiocholine modified peptide FQS*EEQQQTEDELQDK A . The MS 2 spectrum of the triply charged molecular ion of FQS*EEQQQTEDELQDK at m/z 694.983 was obtained with an ESI-LTQ-Orbitrap as described in “Methods”. The fragment ion resulting from the neutral loss of trimethylamine from the parent ion was not detected. Shown in the expanded spectrum is an example of the diagnostic triad consisting of the b 5 + ion resulting from peptide bond cleavage ( m/z =722.3) and further neutral loss of trimethylamine (−59Da, m/z =663.2) or the thiocholine thiolate (−119Da, m/z =603.2). “S*” indicates the thiocholine modified site. B . The MS 3 spectrum of the y 14 +2 ion at m/z 904.4 resulting from the fragmentation of the triply charged molecular ion of FQS*EEQQQTEDELQDK at m/z 694.983. The ion peak at m/z 875.1 corresponds to the doubly charged fragment ion generated from the neutral loss of trimethylamine from the parent ion y 14 +2 . C . The MS 2 spectrum of the singly charged molecular ion of FQS*EEQQQTEDELQDK at m/z 2082.62, obtained with a 4800 MALDI-TOF/TOF Analyzer as described in “Methods”. The ion peak at m/z 2023.72 corresponds to the fragment ion resulting from the neutral loss of trimethylamine from the parent ion. Shown in the expanded spectrum are the low-abundance y ions.

    Techniques Used: Modification, Mass Spectrometry, Diagnostic Assay, Generated

    21) Product Images from "Intestinal epithelial N-acylphosphatidylethanolamine phospholipase D links dietary fat to metabolic adaptations in obesity and steatosis"

    Article Title: Intestinal epithelial N-acylphosphatidylethanolamine phospholipase D links dietary fat to metabolic adaptations in obesity and steatosis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08051-7

    Validation of IEC Napepld deletion. a Napepld mRNA expression in the jejunum, colon, liver and epididymal adipose tissue (EAT) in ND-fed WT and Napepld ∆IEC mice ( n = 8–10). b NAPE-PLD protein levels in the colon. Representative western-blot of NAPE-PLD and β-Actin ( n = 9–10). c Levels of N -acylethanolamines and 2-acylglycerols in small intestinal epithelial cells of ND-fed WT and Napepld ∆IEC mice ( n = 7–9) were determined by using high-performance liquid chromatography-MS using an LTQ Orbitrap mass spectrometer as described in the methods. Dark blue: WT ND mice, light blue: Napepld ∆IEC ND mice. Data are presented as the mean ± s.e.m. *, ** and *** indicate a significant difference versus WT ND (Respectively P
    Figure Legend Snippet: Validation of IEC Napepld deletion. a Napepld mRNA expression in the jejunum, colon, liver and epididymal adipose tissue (EAT) in ND-fed WT and Napepld ∆IEC mice ( n = 8–10). b NAPE-PLD protein levels in the colon. Representative western-blot of NAPE-PLD and β-Actin ( n = 9–10). c Levels of N -acylethanolamines and 2-acylglycerols in small intestinal epithelial cells of ND-fed WT and Napepld ∆IEC mice ( n = 7–9) were determined by using high-performance liquid chromatography-MS using an LTQ Orbitrap mass spectrometer as described in the methods. Dark blue: WT ND mice, light blue: Napepld ∆IEC ND mice. Data are presented as the mean ± s.e.m. *, ** and *** indicate a significant difference versus WT ND (Respectively P

    Techniques Used: Expressing, Mouse Assay, Western Blot, High Performance Liquid Chromatography, Mass Spectrometry

    22) Product Images from "Dynamic metabolic reprogramming in dendritic cells: an early response to influenza infection that is essential for effector function"

    Article Title: Dynamic metabolic reprogramming in dendritic cells: an early response to influenza infection that is essential for effector function

    Journal: bioRxiv

    doi: 10.1101/2020.01.14.906826

    Validation and experimental design of proteomics for IAV infected DC. DC were left untreated (Ctl) or infected for 17 hours at MOI 5 with viable virus (IAV). (A) DC were also infected-propiolactone inactivated virus (IAV BPL ). DC were fixed and stained for DAPI, influenza nuclear protein or murine-enolase protein and visualized with confocal microscopy. (B) Control uninfected cells or IAV infected BMDC (MOI 5 pfu for 17 hours) were separated into soluble and insoluble fractions. The iTRAQ labeled samples were subjected to FASP digestion while the SIL samples received trypsin-catalyzed 18 O/ 16 O labeling. The samples were desalted with C18 SPE, processed with a custom RPLC system and analyzed with a Velos Orbitrap mass spectrometer (iTRAQ) or LTQ-Orbitrap (SIL). (C) Venn-diagram depicting the distinct total number of proteins identified by iTRAQ and SIL, as well as the overlapping number of proteins. Venn-diagram illustrating the statistically significant number of proteins identified by iTRAQ and SIDL in soluble and insoluble fractions which were up and down regulated as well as the overlapping number of proteins. (D) Both soluble and insoluble SIL DC proteomes were submitted to DAVID and PPI spider to define glycolytic protein-protein interaction networks. The glycolytic network was put into Cytoscape and integrated with quantitative data from the proteomic analysis. Alpha enolase increased in soluble and insoluble (inset) proteomes and was validated with immunoblotting revealing the monomer and dimer increased in both soluble (S) and insoluble (I) networks.
    Figure Legend Snippet: Validation and experimental design of proteomics for IAV infected DC. DC were left untreated (Ctl) or infected for 17 hours at MOI 5 with viable virus (IAV). (A) DC were also infected-propiolactone inactivated virus (IAV BPL ). DC were fixed and stained for DAPI, influenza nuclear protein or murine-enolase protein and visualized with confocal microscopy. (B) Control uninfected cells or IAV infected BMDC (MOI 5 pfu for 17 hours) were separated into soluble and insoluble fractions. The iTRAQ labeled samples were subjected to FASP digestion while the SIL samples received trypsin-catalyzed 18 O/ 16 O labeling. The samples were desalted with C18 SPE, processed with a custom RPLC system and analyzed with a Velos Orbitrap mass spectrometer (iTRAQ) or LTQ-Orbitrap (SIL). (C) Venn-diagram depicting the distinct total number of proteins identified by iTRAQ and SIL, as well as the overlapping number of proteins. Venn-diagram illustrating the statistically significant number of proteins identified by iTRAQ and SIDL in soluble and insoluble fractions which were up and down regulated as well as the overlapping number of proteins. (D) Both soluble and insoluble SIL DC proteomes were submitted to DAVID and PPI spider to define glycolytic protein-protein interaction networks. The glycolytic network was put into Cytoscape and integrated with quantitative data from the proteomic analysis. Alpha enolase increased in soluble and insoluble (inset) proteomes and was validated with immunoblotting revealing the monomer and dimer increased in both soluble (S) and insoluble (I) networks.

    Techniques Used: Infection, Staining, Confocal Microscopy, Labeling, Mass Spectrometry

    23) Product Images from "Dok-7 regulates neuromuscular synapse formation by recruiting Crk and Crk-L"

    Article Title: Dok-7 regulates neuromuscular synapse formation by recruiting Crk and Crk-L

    Journal: Genes & Development

    doi: 10.1101/gad.1977710

    Agrin stimulates phosphorylation of Dok-7 Y396 and Y406. ( A ) Dok-7 was immunoprecipitated from lysates of C2 myotubes, which had been stimulated with Agrin for 30 min, or from 293 cells transfected with Dok-7 and MuSK. Following SDS-PAGE, Dok-7 was digested with trypsin, and the tryptic fragments were analyzed by LTQ-Orbitrap MS. Agrin stimulated phosphorylation of Y406 in C2 myotubes; both Y396 and Y406 were phosphorylated in 293 cells transfected with Dok-7 and MuSK. ( B ) Agrin stimulated phosphorylation of Y396 and Y406 in C2 myotubes. ( C ) Western blots of lysates (WCL) from transfected 293 cells show that Dok-7 was not detectably phosphorylated at Y348 or Y355, indicating that Y396 and Y406 are the only tyrosine phosphorylation sites in reconstituted 293 cells ( n = 3; mean ± SEM). ( D ) Y396 and Y406 are the major tyrosine phosphorylation sites in Dok-7 expressed in C2 myotubes. Mutation of either Y396 or Y406 reduced the extent of Agrin-independent Dok-7 phosphorylation. Mutation of both residues reduced phosphorylation to background levels, indicating that Y396 and Y406s are the major, if not only, tyrosine phosphorylation sites. See also Supplemental Figure S1.
    Figure Legend Snippet: Agrin stimulates phosphorylation of Dok-7 Y396 and Y406. ( A ) Dok-7 was immunoprecipitated from lysates of C2 myotubes, which had been stimulated with Agrin for 30 min, or from 293 cells transfected with Dok-7 and MuSK. Following SDS-PAGE, Dok-7 was digested with trypsin, and the tryptic fragments were analyzed by LTQ-Orbitrap MS. Agrin stimulated phosphorylation of Y406 in C2 myotubes; both Y396 and Y406 were phosphorylated in 293 cells transfected with Dok-7 and MuSK. ( B ) Agrin stimulated phosphorylation of Y396 and Y406 in C2 myotubes. ( C ) Western blots of lysates (WCL) from transfected 293 cells show that Dok-7 was not detectably phosphorylated at Y348 or Y355, indicating that Y396 and Y406 are the only tyrosine phosphorylation sites in reconstituted 293 cells ( n = 3; mean ± SEM). ( D ) Y396 and Y406 are the major tyrosine phosphorylation sites in Dok-7 expressed in C2 myotubes. Mutation of either Y396 or Y406 reduced the extent of Agrin-independent Dok-7 phosphorylation. Mutation of both residues reduced phosphorylation to background levels, indicating that Y396 and Y406s are the major, if not only, tyrosine phosphorylation sites. See also Supplemental Figure S1.

    Techniques Used: Immunoprecipitation, Transfection, SDS Page, Mass Spectrometry, Western Blot, Mutagenesis

    24) Product Images from "Policing Starter Unit Selection of the Enterocin Type II Polyketide Synthase by the Type II Thioesterase EncL"

    Article Title: Policing Starter Unit Selection of the Enterocin Type II Polyketide Synthase by the Type II Thioesterase EncL

    Journal: Bioorganic & medicinal chemistry

    doi: 10.1016/j.bmc.2011.04.024

    Qualitative time course of acetyl- and benzoyl-EncC incubated with EncL at room temperature. Shown are the deconvoluted LTQ-orbitrap mass spectra of the reaction mixture before the addition of EncL (−EncL) and at the indicated times after the addition of EncL. Indicated in Da are the theoretical (Calc) and experimentally observed (Obs) monoisotopic masses of holo -EncC, acetyl-EncC, and benzoyl-EncC. The acetyl-loaded form of EncC was depleted already 30 seconds after the addition of EncL with a corresponding increase of holo -EncC. In contrast, benzoyl-EncC is hydrolyzed relatively slowly. The concentration of each acyl-EncC form was ~20 μM, while the concentration of EncL was ~0.5 μM. Some amount of apo -EncC (calculated mass: 12086.3 Da, observed 12086.1 Da) was clearly visible (vertical arrow) in the control sample without EncL but not in the other samples, probably because of phosphopanteteinylation of apo -EncC by Sfp with residual acyl-CoA. Masses of other strong bands visible in the spectra are consistent with phosphate adducts (+98 Da), potassium adducts (+38), and oxidized forms (+16) of the main species indicated, as shown by the horizontal arrows.
    Figure Legend Snippet: Qualitative time course of acetyl- and benzoyl-EncC incubated with EncL at room temperature. Shown are the deconvoluted LTQ-orbitrap mass spectra of the reaction mixture before the addition of EncL (−EncL) and at the indicated times after the addition of EncL. Indicated in Da are the theoretical (Calc) and experimentally observed (Obs) monoisotopic masses of holo -EncC, acetyl-EncC, and benzoyl-EncC. The acetyl-loaded form of EncC was depleted already 30 seconds after the addition of EncL with a corresponding increase of holo -EncC. In contrast, benzoyl-EncC is hydrolyzed relatively slowly. The concentration of each acyl-EncC form was ~20 μM, while the concentration of EncL was ~0.5 μM. Some amount of apo -EncC (calculated mass: 12086.3 Da, observed 12086.1 Da) was clearly visible (vertical arrow) in the control sample without EncL but not in the other samples, probably because of phosphopanteteinylation of apo -EncC by Sfp with residual acyl-CoA. Masses of other strong bands visible in the spectra are consistent with phosphate adducts (+98 Da), potassium adducts (+38), and oxidized forms (+16) of the main species indicated, as shown by the horizontal arrows.

    Techniques Used: Incubation, Concentration Assay

    25) Product Images from "Proteomic profiling of KATP channel-deficient hypertensive heart maps risk for maladaptive cardiomyopathic outcome"

    Article Title: Proteomic profiling of KATP channel-deficient hypertensive heart maps risk for maladaptive cardiomyopathic outcome

    Journal: Proteomics

    doi: 10.1002/pmic.200800718

    Metabolic enzymes associated with K ATP channel function are among the differentially affected subproteome. (A) Representative LTQ-Orbitrap MS/MS product ion spectra obtained for spots #40 (upper) and #67 (lower), modified from BioWorks 3.2 to indicate
    Figure Legend Snippet: Metabolic enzymes associated with K ATP channel function are among the differentially affected subproteome. (A) Representative LTQ-Orbitrap MS/MS product ion spectra obtained for spots #40 (upper) and #67 (lower), modified from BioWorks 3.2 to indicate

    Techniques Used: Mass Spectrometry, Modification

    Metabolism is the predominant function of Kir6.2-KO proteins differentially altered in response to HTN. Significantly altered spots identified by LTQ-Orbitrap MS/MS analysis and functionally categorized by their respective Swiss-Prot ontological annotations
    Figure Legend Snippet: Metabolism is the predominant function of Kir6.2-KO proteins differentially altered in response to HTN. Significantly altered spots identified by LTQ-Orbitrap MS/MS analysis and functionally categorized by their respective Swiss-Prot ontological annotations

    Techniques Used: Mass Spectrometry

    Remaining Kir6.2-KO proteins differentially altered in response to HTN comprise a metabolism infrastructure related module. Significantly altered spots identified by LTQ-Orbitrap MS/MS analysis included 41 proteins, and functionally categorized by their
    Figure Legend Snippet: Remaining Kir6.2-KO proteins differentially altered in response to HTN comprise a metabolism infrastructure related module. Significantly altered spots identified by LTQ-Orbitrap MS/MS analysis included 41 proteins, and functionally categorized by their

    Techniques Used: Mass Spectrometry

    26) Product Images from "The Role of Methoxy Group in the Nazarov Cyclization of 1,5-bis-(2-Methoxyphenyl)-1,4-Pentadien-3-one in the Gas Phase and Condensed Phase"

    Article Title: The Role of Methoxy Group in the Nazarov Cyclization of 1,5-bis-(2-Methoxyphenyl)-1,4-Pentadien-3-one in the Gas Phase and Condensed Phase

    Journal: Journal of the American Society for Mass Spectrometry

    doi: 10.1007/s13361-013-0785-8

    The CAD mass spectra of the [M + H] + ions of Compounds ( a ) 3 , ( b ) 4 , and ( c ) 5 ; from the Thermo LTQ Orbitrap mass spectrometer
    Figure Legend Snippet: The CAD mass spectra of the [M + H] + ions of Compounds ( a ) 3 , ( b ) 4 , and ( c ) 5 ; from the Thermo LTQ Orbitrap mass spectrometer

    Techniques Used: Mass Spectrometry

    Positive-ion ESI CAD mass spectra of ( a ) bis -(2-methoxybenzal)acetone ( 1 ), ( b ) 2,3- bis -(2-methoxyphenyl)-cyclopent-2-ene-1-one ( 2 ): Thermo LTQ Orbitrap mass spectrometer
    Figure Legend Snippet: Positive-ion ESI CAD mass spectra of ( a ) bis -(2-methoxybenzal)acetone ( 1 ), ( b ) 2,3- bis -(2-methoxyphenyl)-cyclopent-2-ene-1-one ( 2 ): Thermo LTQ Orbitrap mass spectrometer

    Techniques Used: Mass Spectrometry

    27) Product Images from "Improved LC-MS/MS Spectral Counting Statistics by Recovering Low Scoring Spectra Matched to Confidently Identified Peptide Sequences"

    Article Title: Improved LC-MS/MS Spectral Counting Statistics by Recovering Low Scoring Spectra Matched to Confidently Identified Peptide Sequences

    Journal: Journal of proteome research

    doi: 10.1021/pr100508p

    Recovered spectra have similar LC retention time with high scored spectra The average scan numbers of recovered spectra of all individual peptides are plotted against filtered spectra. Peptides with RSD of filtered scan number > 20% are not included. Both datasets are collected from single LC-MS/MS runs. (A), data from Orbitrap. (B), data from LTQ.
    Figure Legend Snippet: Recovered spectra have similar LC retention time with high scored spectra The average scan numbers of recovered spectra of all individual peptides are plotted against filtered spectra. Peptides with RSD of filtered scan number > 20% are not included. Both datasets are collected from single LC-MS/MS runs. (A), data from Orbitrap. (B), data from LTQ.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    28) Product Images from "Proteomic Analysis Reveals Warburg Effect and Anomalous Metabolism of Glutamine in Pancreatic Cancer Cells"

    Article Title: Proteomic Analysis Reveals Warburg Effect and Anomalous Metabolism of Glutamine in Pancreatic Cancer Cells

    Journal: Journal of proteome research

    doi: 10.1021/pr2009274

    Chromatogram of LC–MS/MS analysis of tryptic peptides from normal pancreatic duct and PANC-1 cells. Twenty microgram tryptic peptides and 100 fmol standard peptide Ang I were loaded to C 18 capillary column, and the eluted peptides were analyzed by LTQ-Orbitrap. For the first iteration of normal duct sample, the retention time of exogenous control Ang I was 78 min, and the retention times of 4 internal control peptides were 26 min, 42 min, 56 min, and 91 min respectively. Labeled peak A is the peptide IWHHTFYNELR (2+ ion, m / z 758.3766) from actin, gamma 1; peak B is the peptide VAPEEHPVLLTEAPLNPK (2+ ion, m / z 977.5364) from actin, gamma 1; peak C is the peptide LGEHNIDVLEGNEQFINAAK (2+ ion, m / z 1106.0545) from trypsin autolysis; peak D is the peptide LCYVALDFEQEMATAASSSSLEK (2+ ion, m / z 1275.5953) from actin, gamma 1. The mass accuracy of these peptides was within 5 ppm. These peptides were also identified in the second and third iterations of normal duct sample, and in 3 iterations of PANC-1 sample with similar retention times (±2.0 min), indicating that the experimental procedure was reproducible and the MS results could be compared.
    Figure Legend Snippet: Chromatogram of LC–MS/MS analysis of tryptic peptides from normal pancreatic duct and PANC-1 cells. Twenty microgram tryptic peptides and 100 fmol standard peptide Ang I were loaded to C 18 capillary column, and the eluted peptides were analyzed by LTQ-Orbitrap. For the first iteration of normal duct sample, the retention time of exogenous control Ang I was 78 min, and the retention times of 4 internal control peptides were 26 min, 42 min, 56 min, and 91 min respectively. Labeled peak A is the peptide IWHHTFYNELR (2+ ion, m / z 758.3766) from actin, gamma 1; peak B is the peptide VAPEEHPVLLTEAPLNPK (2+ ion, m / z 977.5364) from actin, gamma 1; peak C is the peptide LGEHNIDVLEGNEQFINAAK (2+ ion, m / z 1106.0545) from trypsin autolysis; peak D is the peptide LCYVALDFEQEMATAASSSSLEK (2+ ion, m / z 1275.5953) from actin, gamma 1. The mass accuracy of these peptides was within 5 ppm. These peptides were also identified in the second and third iterations of normal duct sample, and in 3 iterations of PANC-1 sample with similar retention times (±2.0 min), indicating that the experimental procedure was reproducible and the MS results could be compared.

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

    29) Product Images from "Simultaneous Qualitative Assessment and Quantitative Analysis of Metabolites (Phenolics, Nucleosides and Amino Acids) from the Roots of Fresh Gastrodia elata Using UPLC-ESI-Triple Quadrupole Ion MS and ESI- Linear Ion Trap High-Resolution MS"

    Article Title: Simultaneous Qualitative Assessment and Quantitative Analysis of Metabolites (Phenolics, Nucleosides and Amino Acids) from the Roots of Fresh Gastrodia elata Using UPLC-ESI-Triple Quadrupole Ion MS and ESI- Linear Ion Trap High-Resolution MS

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150647

    Optimized HPLC chromatograms at 220 nm selected from three column system (AI—AIII) and three mobile protocols (BI-BIII). Thirty-one compounds (peaks 1–31) were separated by BIII and identified using UPLC- LTQ Orbitrap mass spectrometry.
    Figure Legend Snippet: Optimized HPLC chromatograms at 220 nm selected from three column system (AI—AIII) and three mobile protocols (BI-BIII). Thirty-one compounds (peaks 1–31) were separated by BIII and identified using UPLC- LTQ Orbitrap mass spectrometry.

    Techniques Used: High Performance Liquid Chromatography, Mass Spectrometry

    30) Product Images from "Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags"

    Article Title: Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags

    Journal: Analytical chemistry

    doi: 10.1021/ac702328x

    The UStags process for identifying unique peptide sequences from precise LC-MS and MS/MS experiments. Yeast whole cell tryptic digest was analyzed using an LC-LTQ-Orbitrap platform; resulting LC-MS/MS dataset was searched using SEQUEST against yeast sequence database. Amino acid residue sequencing (see Supplementary Methods) was applied to obtain accurate AA residue assignments and sequences. These initial unique sequences were examined using the residue replacement filter (RRF; see Supplementary Methods), illustrated here for TSGTLEMNLK(1). This sequence was found in a single protein (the number in the parenthesis); however, an isobaric variant sequence SSGTLEMNLK potentially exists in another yeast protein when modifications are considered (i.e., Ser-me-ester has the same mass as Thr). Due to the ambiguity this sequence cannot serve as an UStag (i.e., only sequences remaining after the RRF are considered UStags).
    Figure Legend Snippet: The UStags process for identifying unique peptide sequences from precise LC-MS and MS/MS experiments. Yeast whole cell tryptic digest was analyzed using an LC-LTQ-Orbitrap platform; resulting LC-MS/MS dataset was searched using SEQUEST against yeast sequence database. Amino acid residue sequencing (see Supplementary Methods) was applied to obtain accurate AA residue assignments and sequences. These initial unique sequences were examined using the residue replacement filter (RRF; see Supplementary Methods), illustrated here for TSGTLEMNLK(1). This sequence was found in a single protein (the number in the parenthesis); however, an isobaric variant sequence SSGTLEMNLK potentially exists in another yeast protein when modifications are considered (i.e., Ser-me-ester has the same mass as Thr). Due to the ambiguity this sequence cannot serve as an UStag (i.e., only sequences remaining after the RRF are considered UStags).

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

    31) Product Images from "Monte Carlo Simulation-Based Algorithms for Analysis of Shotgun Proteomic Data"

    Article Title: Monte Carlo Simulation-Based Algorithms for Analysis of Shotgun Proteomic Data

    Journal:

    doi: 10.1021/pr800002u

    Distributions of pp2 MCS score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap
    Figure Legend Snippet: Distributions of pp2 MCS score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap

    Techniques Used: Mass Spectrometry

    ROC curves of pp2 MCS and pp tag from the MCS-based score models in MassMatrix along with other statistical scores from Mascot, OMSSA and X!Tandem for (a) the LCQ data set and (b) the LTQ-Orbitrap data set.
    Figure Legend Snippet: ROC curves of pp2 MCS and pp tag from the MCS-based score models in MassMatrix along with other statistical scores from Mascot, OMSSA and X!Tandem for (a) the LCQ data set and (b) the LTQ-Orbitrap data set.

    Techniques Used:

    Search speed in terms of MS/MS spectra per second for MassMatrix, Mascot, OMSSA, and X!Tandem on a PC with an Intel quad core CPU (2.4 GHz) and Linux operating system. The two merged data sets from LCQ and LTQ-Orbitrap mass spectrometers were searched
    Figure Legend Snippet: Search speed in terms of MS/MS spectra per second for MassMatrix, Mascot, OMSSA, and X!Tandem on a PC with an Intel quad core CPU (2.4 GHz) and Linux operating system. The two merged data sets from LCQ and LTQ-Orbitrap mass spectrometers were searched

    Techniques Used: Mass Spectrometry

    ROC curves of different score standards in MassMatrix for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap mass spectrometer.
    Figure Legend Snippet: ROC curves of different score standards in MassMatrix for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap mass spectrometer.

    Techniques Used: Mass Spectrometry

    Distributions of pp tag score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap
    Figure Legend Snippet: Distributions of pp tag score from the MCS-based score models for peptides with (a) all charges; (b) +1 charges; and (c) +2/+3 charges from LCQ mass spectrometer and peptides with (d) all charges; (e) +1 charges; and (f) +2/+3 charges from LTQ-Orbitrap

    Techniques Used: Mass Spectrometry

    32) Product Images from "Kinetic Study of Neuropeptide Y (NPY) Proteolysis in Blood and Identification of NPY3–35"

    Article Title: Kinetic Study of Neuropeptide Y (NPY) Proteolysis in Blood and Identification of NPY3–35

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.035253

    Mass spectrometry characterization of NPY 3–35 . A , LTQ Orbitrap, full-MS spectra m / z 350–2000; signals of NPY 3–35 , NPY 3–36 , and NPY 2–36 (charge states +3, +4, and +5 are in bold ). B , LC-MS chromatograms for the most
    Figure Legend Snippet: Mass spectrometry characterization of NPY 3–35 . A , LTQ Orbitrap, full-MS spectra m / z 350–2000; signals of NPY 3–35 , NPY 3–36 , and NPY 2–36 (charge states +3, +4, and +5 are in bold ). B , LC-MS chromatograms for the most

    Techniques Used: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    33) Product Images from "The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1"

    Article Title: The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005811

    PP2C38 is phosphorylated in response to PAMP perception. (A) PAMP perception induces PP2C38 band shift. PPC2C38-FLAG protein expressed in N . benthamiana leaves and treated (+) or not (-) with 100 nM elf18 for the indicated times. Upper band corresponds to phosphorylated PP2C38 form (pPP2C38). 12% bisacrylamide gels were used for better protein separation. Experiment repeated two times with similar results. (B) PP2C band shift is due to phosphorylation. Immunoprecipitated PP2C38-FLAG proteins from N . benthamiana leaves co-expressing EFR-GFP treated with 100 nM elf18 were incubated with calf intestine phosphatase (CIP) in the presence or absence of the phosphatase inhibitor NaF. (C) PP2C38 is phosphorylated on S77 in vivo . Immunoprecipitated PP2C38-FLAG proteins from N . benthamiana were submitted for LTQ-Orbitrap MS/MS analysis. The DpSGPQATFVGVY phosphopeptide was identified as a doubly charged precursor (m/z 660.78) with fragmentation pattern consisting of singly and doubly charged b- and y- ions. Modified peptide sequence and fragmentation pattern shown above spectrum. (D) S77 phosphorylation is required for BIK1 trans-phosphorylation of PP2C38 in vitro . Recombinant GST-BIK1 was incubated with [ 32 P]γ-ATP to promote auto-phosphorylation, followed by addition of recombinant MBP-PP2C38. In vitro PP2C38 trans-phosphorylation is revealed by autoradiography. CBB staining shown as loading control. Experiment repeated two times with similar results. (E-F) S77 is required for flg22-induced PP2C38 band shift. Phospho-dead PP2C38 S77A -FLAG variant transiently expressed in N . benthamiana (E) or in Arabidopsis Col-0 protoplasts (F) does not exhibit a band shift after 20 min 100 nM flg22 treatment. 12% bisacrylamide gels were used for better protein separation. Experiments repeated at least three times with similar results.
    Figure Legend Snippet: PP2C38 is phosphorylated in response to PAMP perception. (A) PAMP perception induces PP2C38 band shift. PPC2C38-FLAG protein expressed in N . benthamiana leaves and treated (+) or not (-) with 100 nM elf18 for the indicated times. Upper band corresponds to phosphorylated PP2C38 form (pPP2C38). 12% bisacrylamide gels were used for better protein separation. Experiment repeated two times with similar results. (B) PP2C band shift is due to phosphorylation. Immunoprecipitated PP2C38-FLAG proteins from N . benthamiana leaves co-expressing EFR-GFP treated with 100 nM elf18 were incubated with calf intestine phosphatase (CIP) in the presence or absence of the phosphatase inhibitor NaF. (C) PP2C38 is phosphorylated on S77 in vivo . Immunoprecipitated PP2C38-FLAG proteins from N . benthamiana were submitted for LTQ-Orbitrap MS/MS analysis. The DpSGPQATFVGVY phosphopeptide was identified as a doubly charged precursor (m/z 660.78) with fragmentation pattern consisting of singly and doubly charged b- and y- ions. Modified peptide sequence and fragmentation pattern shown above spectrum. (D) S77 phosphorylation is required for BIK1 trans-phosphorylation of PP2C38 in vitro . Recombinant GST-BIK1 was incubated with [ 32 P]γ-ATP to promote auto-phosphorylation, followed by addition of recombinant MBP-PP2C38. In vitro PP2C38 trans-phosphorylation is revealed by autoradiography. CBB staining shown as loading control. Experiment repeated two times with similar results. (E-F) S77 is required for flg22-induced PP2C38 band shift. Phospho-dead PP2C38 S77A -FLAG variant transiently expressed in N . benthamiana (E) or in Arabidopsis Col-0 protoplasts (F) does not exhibit a band shift after 20 min 100 nM flg22 treatment. 12% bisacrylamide gels were used for better protein separation. Experiments repeated at least three times with similar results.

    Techniques Used: Electrophoretic Mobility Shift Assay, Immunoprecipitation, Expressing, Incubation, In Vivo, Mass Spectrometry, Modification, Sequencing, In Vitro, Recombinant, Autoradiography, Staining, Variant Assay

    34) Product Images from "ATP-Sensitive K+ Channel Knockout Induces Cardiac Proteome Remodeling Predictive of Heart Disease Susceptibility"

    Article Title: ATP-Sensitive K+ Channel Knockout Induces Cardiac Proteome Remodeling Predictive of Heart Disease Susceptibility

    Journal: Journal of proteome research

    doi: 10.1021/pr900561g

    Metabolic remodeling is the primary effect of K ATP channel deficiency. Proteins assigned by LTQ-Orbitrap MS/MS analysis from significantly altered spots were functionally categorized by Swiss-Prot ontological annotations. The primary association was with
    Figure Legend Snippet: Metabolic remodeling is the primary effect of K ATP channel deficiency. Proteins assigned by LTQ-Orbitrap MS/MS analysis from significantly altered spots were functionally categorized by Swiss-Prot ontological annotations. The primary association was with

    Techniques Used: Mass Spectrometry

    35) Product Images from "Shotgun Lipidomics Identifies a Paired Rule for the Presence of Isomeric Ether Phospholipid Molecular Species"

    Article Title: Shotgun Lipidomics Identifies a Paired Rule for the Presence of Isomeric Ether Phospholipid Molecular Species

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001368

    Representative negative-ion ESI/MS analyses of bovine heart ethanolamine glycerophospholipid molecular species. Bovine heart lipids were extracted by a modified Bligh and Dyer procedure [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23] . Analyses of PtdEtn molecular species were performed in the negative-ion mode by using an LTQ-Orbitrap mass spectrometer equipped with a Nanomate device as described under “ MATERIALS AND METHODS ”.
    Figure Legend Snippet: Representative negative-ion ESI/MS analyses of bovine heart ethanolamine glycerophospholipid molecular species. Bovine heart lipids were extracted by a modified Bligh and Dyer procedure [21] and the PtdEtn fraction was separated by using HPLC with a cation-exchange column as previously described [23] . Analyses of PtdEtn molecular species were performed in the negative-ion mode by using an LTQ-Orbitrap mass spectrometer equipped with a Nanomate device as described under “ MATERIALS AND METHODS ”.

    Techniques Used: Mass Spectrometry, Modification, High Performance Liquid Chromatography

    Product ion analyses of synthetic 18∶0-20∶4 plasmenylethanolamine molecular species in the negative-ion mode. Product ion ESI/MS analysis of deprotonated 18∶0-20∶4 plasmenylethanolamine at m/z 750.54 was performed on an LTQ-Orbitrap mass spectrometer with a C-trap using an ion selective window of 1 Th by LTQ. Collision activation in C-trap was carried out with normalized collision energy of 55% and gas pressure of 1 mT. The resultant fragment ions were analyzed in the Orbitrap. The arrow indicates the absence of the 18:0 FA carboxylate in the spectrum after amplifying the position greater than1,000 fold.
    Figure Legend Snippet: Product ion analyses of synthetic 18∶0-20∶4 plasmenylethanolamine molecular species in the negative-ion mode. Product ion ESI/MS analysis of deprotonated 18∶0-20∶4 plasmenylethanolamine at m/z 750.54 was performed on an LTQ-Orbitrap mass spectrometer with a C-trap using an ion selective window of 1 Th by LTQ. Collision activation in C-trap was carried out with normalized collision energy of 55% and gas pressure of 1 mT. The resultant fragment ions were analyzed in the Orbitrap. The arrow indicates the absence of the 18:0 FA carboxylate in the spectrum after amplifying the position greater than1,000 fold.

    Techniques Used: Mass Spectrometry, Activation Assay

    Product ion analyses of individual molecular species present in isolated bovine heart ethanolamine glycerophospholipids. Product ion ESI/MS analyses of PtdEtn molecular species in isolated bovine heart ethanolamine glycerophospholipids at m/z 772.5 (Panel A) and 774.5 (Panel B) were performed by using an LTQ-Orbitrap mass spectrometer. Analyses were conducted using a peak width setting of 1 Th by selection of the molecular ion in the linear ion trap (LTQ), collision activation in the C-trap with a normalized collision energy of 55% and gas pressure of 1 mTorr, and analysis of the resultant product ions in the Orbitrap.
    Figure Legend Snippet: Product ion analyses of individual molecular species present in isolated bovine heart ethanolamine glycerophospholipids. Product ion ESI/MS analyses of PtdEtn molecular species in isolated bovine heart ethanolamine glycerophospholipids at m/z 772.5 (Panel A) and 774.5 (Panel B) were performed by using an LTQ-Orbitrap mass spectrometer. Analyses were conducted using a peak width setting of 1 Th by selection of the molecular ion in the linear ion trap (LTQ), collision activation in the C-trap with a normalized collision energy of 55% and gas pressure of 1 mTorr, and analysis of the resultant product ions in the Orbitrap.

    Techniques Used: Isolation, Mass Spectrometry, Selection, Activation Assay

    36) Product Images from "Establishing a protein expression profile database for the normal human pituitary gland using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry ☆"

    Article Title: Establishing a protein expression profile database for the normal human pituitary gland using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry ☆

    Journal: Neural Regeneration Research

    doi: 10.3969/j.issn.1673-5374.2012.36.010

    Block diagram of protein separation and identification using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap (collagen as an example). (A) Total ion-current in chromatography with 100 mM ammonium acetate solution. The horizontal axis represents wash speed and the vertical axis represents relative intensity. (B) The spectrum signal for the collagen protein changed with time. (C) The chromatographic peak of one peptide of the collagen protein. (B, C) The horizontal axis represents time and the vertical axis represents the relative abundance of the protein. (D) Process of protein identification using software. The top line of letters represents the identified amino acid. Below, at t = 64.15 minutes, is the mass chromatogram for the collagen peptides. (E) The composition of the amino acids of the collagen protein. The yellow area represents the functional domain of the protein.
    Figure Legend Snippet: Block diagram of protein separation and identification using two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap (collagen as an example). (A) Total ion-current in chromatography with 100 mM ammonium acetate solution. The horizontal axis represents wash speed and the vertical axis represents relative intensity. (B) The spectrum signal for the collagen protein changed with time. (C) The chromatographic peak of one peptide of the collagen protein. (B, C) The horizontal axis represents time and the vertical axis represents the relative abundance of the protein. (D) Process of protein identification using software. The top line of letters represents the identified amino acid. Below, at t = 64.15 minutes, is the mass chromatogram for the collagen peptides. (E) The composition of the amino acids of the collagen protein. The yellow area represents the functional domain of the protein.

    Techniques Used: Blocking Assay, High Performance Liquid Chromatography, Chromatography, Software, Functional Assay

    Expression of vimentin and carbonic anhydrase I in normal pituitary tissue. Western blot assay results showing high expression of vimentin and carbonic anhydrase I (CAI) in two normal pituitary samples (lanes 2 and 3) and low expression in normal brain tissue (lane 1). Beta-actin was used as an internal standard. Our findings were consistent with those from two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry. Molecular weights are shown at the left.
    Figure Legend Snippet: Expression of vimentin and carbonic anhydrase I in normal pituitary tissue. Western blot assay results showing high expression of vimentin and carbonic anhydrase I (CAI) in two normal pituitary samples (lanes 2 and 3) and low expression in normal brain tissue (lane 1). Beta-actin was used as an internal standard. Our findings were consistent with those from two-dimensional high-performance liquid chromatography combined with LTQ-Orbitrap mass spectrometry. Molecular weights are shown at the left.

    Techniques Used: Expressing, Western Blot, High Performance Liquid Chromatography, Mass Spectrometry

    37) Product Images from "Simultaneous Qualitation and Quantitation of Chlorogenic Acids in Kuding Tea Using Ultra-High-Performance Liquid Chromatography–Diode Array Detection Coupled with Linear Ion Trap–Orbitrap Mass Spectrometer"

    Article Title: Simultaneous Qualitation and Quantitation of Chlorogenic Acids in Kuding Tea Using Ultra-High-Performance Liquid Chromatography–Diode Array Detection Coupled with Linear Ion Trap–Orbitrap Mass Spectrometer

    Journal: Molecules

    doi: 10.3390/molecules21121728

    UHPLC-DAD-LTQ-Orbitrap analysis of CGAs in Kuding tea (2 μL): UHPLC-DAD chromatogram of reference standards ( A ) and the extract ( B ) at 327 nm; the total ion chromatogram (TIC) of reference standards ( C ) and the extract ( D ) in negative mode.
    Figure Legend Snippet: UHPLC-DAD-LTQ-Orbitrap analysis of CGAs in Kuding tea (2 μL): UHPLC-DAD chromatogram of reference standards ( A ) and the extract ( B ) at 327 nm; the total ion chromatogram (TIC) of reference standards ( C ) and the extract ( D ) in negative mode.

    Techniques Used:

    38) Product Images from "Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica"

    Article Title: Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0143113

    Functional annotation of the microsomal proteins present in the JMY1877 transformants. Proteins in 100,000 g microsomes from three independent transformants of the control strain or the EgDGAT1-1 expressing strain were digested in gel. Peptides were separated by liquid chromatography and analyzed with a LTQ Orbitrap mass spectrometer using a nano-electrospray interface. Proteins found in the three transformants of each strain were annotated using the Génolevures annotated sequence database [ 39 ] and manually curated into twelve functional classes. Six hundred and twenty-five of the 744 proteins could be sorted into twelve functional classes.
    Figure Legend Snippet: Functional annotation of the microsomal proteins present in the JMY1877 transformants. Proteins in 100,000 g microsomes from three independent transformants of the control strain or the EgDGAT1-1 expressing strain were digested in gel. Peptides were separated by liquid chromatography and analyzed with a LTQ Orbitrap mass spectrometer using a nano-electrospray interface. Proteins found in the three transformants of each strain were annotated using the Génolevures annotated sequence database [ 39 ] and manually curated into twelve functional classes. Six hundred and twenty-five of the 744 proteins could be sorted into twelve functional classes.

    Techniques Used: Functional Assay, Expressing, Liquid Chromatography, Mass Spectrometry, Sequencing

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    Article Title: Discovering and validating unknown phospho-sites from p38 and HuR protein kinases in vitro by Phosphoproteomic and Bioinformatic tools
    Article Snippet: .. In fact, multistage activation resulted in more information for the suite of phosphopeptides studied (Table ) (see an example of the spectrum of an identified phosphorylated peptide when using SIMAC coupled to MSA in the LTQ ion Trap mass spectrometer and Mascot, Figure ). ..

    Mass Spectrometry:

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    Article Title: Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex
    Article Snippet: .. Individual TiO2 -bound phosphopeptide fractions were analyzed by multi-dimensional LC-MS/MS on an LTQ linear ion trap mass spectrometer (Thermo Scientific), according to published protocols . ..

    Article Title: High Throughput Characterization of Combinatorial Histone Codes *
    Article Snippet: .. Several other buffer systems for the B mobile phase were tried during method development as noted under “Results.” The column eluent was introduced into an LTQ-ETD ion trap mass spectrometer (Thermo Scientific, Waltham, MA) or an LTQ-Orbitrap XL (Thermo Scientific) (data in only) by nanoelectrospray ionization. .. Every cycle a full mass spectrum was acquired from 300 to 2000 m/z followed by narrower mass range full mass spectrum to select a given charge state of the histone for data-dependent selection.

    Article Title: Systematic metabolic profiling and bioactivity assays for bioconversion of Aceraceae family
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    Thermo Fisher ltq orbitrap mass spectrometer
    Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an <t>LTQ-Orbitrap</t> mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.
    Ltq Orbitrap Mass Spectrometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 3725 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher ltq orbitrap velos mass spectrometer
    A schematic workflow illustrating the steps involved in the differential analysis of RA and OA synovial fluid proteome. Proteins from RA and OA synovial fluid were extracted and depleted to remove the 14 most abundant proteins using multiple affinity removal system, Human-14. The depleted protein from RA and OA were then digested with trypsin and labeled with iTRAQ reagents, 117 and 116 respectively. The labeled samples were pooled and subjected to fractionation using strong cation exchange chromatography. The fractions were then analyzed on a <t>LTQ-Orbitrap</t> <t>Velos</t> mass spectrometer. The MS/MS data obtained was searched against Human RefSeq 50 database using Sequest and Mascot search algorithms. Validation of the iTRAQ quantitation data was carried out using multiple reaction monitoring and Western blot.
    Ltq Orbitrap Velos Mass Spectrometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.

    Journal: Open Biology

    Article Title: PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

    doi: 10.1098/rsob.120080

    Figure Lengend Snippet: Human Parkin Ser 65 is a substrate of human PINK1 upon CCCP stimulation. ( a ) Confirmation by mass spectrometry that Ser 65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser 131 and Ser 65 phosphopeptide (3 + R.NDWTVQNCDLDQQ S IVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y -axis and retention time is plotted on the x -axis. The m / z value corresponding to the Ser 131 phosphopeptide was detected in all conditions whilst that of the Ser 65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. ( b ) Characterization of Parkin phospho-Ser 65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser 65 Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser 65 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.

    Article Snippet: Isolated phosphopeptides were analysed by LC-MS-MS on a proxeon Easy-nLC nano liquid chromatography system coupled to a Thermo LTQ-orbitrap mass spectrometer.

    Techniques: Mass Spectrometry, Activation Assay, Expressing, Transfection, Lysis, Immunoprecipitation, SDS Page, Staining, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mutagenesis

    Schematic diagram of proteomic analysis of the mouse organ of Corti (OC) sample. ( 1 ) The OCs were reconstituted in 100 μL lysis buffer (100 mM ammonium bicarbonate, pH 8.4); ( 2 ) The lysates were reduced by 5 mM DL-Dithiothreitol (DTT) and digested by trypsin overnight; ( 3 ) The digests were desalted and dried in a vacuum centrifuge immediately after digestion; ( 4 ) Dried peptides were subjected to the in-house assembled reverse phase metal-free multiple-column nanoLC system coupled with ( 5 ) LTQ-Orbitrap XL mass spectrometer for MS analysis.

    Journal: International Journal of Molecular Sciences

    Article Title: Proteomic Analysis of the Organ of Corti Using Nanoscale Liquid Chromatography Coupled with Tandem Mass Spectrometry

    doi: 10.3390/ijms13078171

    Figure Lengend Snippet: Schematic diagram of proteomic analysis of the mouse organ of Corti (OC) sample. ( 1 ) The OCs were reconstituted in 100 μL lysis buffer (100 mM ammonium bicarbonate, pH 8.4); ( 2 ) The lysates were reduced by 5 mM DL-Dithiothreitol (DTT) and digested by trypsin overnight; ( 3 ) The digests were desalted and dried in a vacuum centrifuge immediately after digestion; ( 4 ) Dried peptides were subjected to the in-house assembled reverse phase metal-free multiple-column nanoLC system coupled with ( 5 ) LTQ-Orbitrap XL mass spectrometer for MS analysis.

    Article Snippet: The nanoLC system was interfaced with a LTQ-Orbitrap XL mass spectrometer equipped with a nanoelectrospray ion source (Thermo Scientific, Waltham, MA, USA).

    Techniques: Lysis, Mass Spectrometry

    Quantification of calcium-dependent regulation of phosphorylation sites of Toxoplasma invasion motor complex components. A ) Work flow to identify individual phosphorylation sites and quantitatively assess their responsiveness to calcium signals using a SILAC-based proteomics approach. A 1∶1 mixture of Triton X-100 lysates from “Heavy” (H; Arg4/Lys8)-labeled ethanol-stimulated tachyzoites or

    Journal: PLoS Pathogens

    Article Title: Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

    doi: 10.1371/journal.ppat.1002222

    Figure Lengend Snippet: Quantification of calcium-dependent regulation of phosphorylation sites of Toxoplasma invasion motor complex components. A ) Work flow to identify individual phosphorylation sites and quantitatively assess their responsiveness to calcium signals using a SILAC-based proteomics approach. A 1∶1 mixture of Triton X-100 lysates from “Heavy” (H; Arg4/Lys8)-labeled ethanol-stimulated tachyzoites or "Light" (L; Arg0/Lys0)-labeled non-stimulated parasites was generated, and a TiO 2 -enriched phosphopeptide sample of H/L-labeled Toxoplasma invasion motor complexes was prepared and analysed by LC-MS/MS on an LTQ-Orbitrap instrument. Mascot and MaxQuant search engines facilitated subsequent manual identification, phosphosite localization and quantification of proteins or peptides as detailed in materials and mathods. B ) Sypro Ruby-stained SDS-PAGE separation of the relative amounts of light (lane 1) or heavy (lane 2) Triton X-100 whole protein extracts are shown. Intact tachyzoite invasion motor complexes comprising the five major components MyoA, GAP50, GAP45 and MLC1 were precipitated from a 1∶1 H/L mixture by GAP45-specific immuno-affinity chromatography (lane 3).

    Article Snippet: The nano HPLC was coupled on-line to an LTQ-Orbitrap mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific) for automated MS/MS.

    Techniques: Flow Cytometry, Labeling, Generated, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Staining, SDS Page, Affinity Chromatography

    A schematic workflow illustrating the steps involved in the differential analysis of RA and OA synovial fluid proteome. Proteins from RA and OA synovial fluid were extracted and depleted to remove the 14 most abundant proteins using multiple affinity removal system, Human-14. The depleted protein from RA and OA were then digested with trypsin and labeled with iTRAQ reagents, 117 and 116 respectively. The labeled samples were pooled and subjected to fractionation using strong cation exchange chromatography. The fractions were then analyzed on a LTQ-Orbitrap Velos mass spectrometer. The MS/MS data obtained was searched against Human RefSeq 50 database using Sequest and Mascot search algorithms. Validation of the iTRAQ quantitation data was carried out using multiple reaction monitoring and Western blot.

    Journal: Clinical proteomics

    Article Title: Differential proteomic analysis of synovial fluid from rheumatoid arthritis and osteoarthritis patients

    doi: 10.1186/1559-0275-11-1

    Figure Lengend Snippet: A schematic workflow illustrating the steps involved in the differential analysis of RA and OA synovial fluid proteome. Proteins from RA and OA synovial fluid were extracted and depleted to remove the 14 most abundant proteins using multiple affinity removal system, Human-14. The depleted protein from RA and OA were then digested with trypsin and labeled with iTRAQ reagents, 117 and 116 respectively. The labeled samples were pooled and subjected to fractionation using strong cation exchange chromatography. The fractions were then analyzed on a LTQ-Orbitrap Velos mass spectrometer. The MS/MS data obtained was searched against Human RefSeq 50 database using Sequest and Mascot search algorithms. Validation of the iTRAQ quantitation data was carried out using multiple reaction monitoring and Western blot.

    Article Snippet: LC-MS/MS analysis Tandem mass spectrometric analysis of the iTRAQ labeled peptides were carried out using LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, Bremen, Germany) interfaced with Easy nanoLC II (previously Proxeon, Thermo Scientific, Bremen, Germany).

    Techniques: Labeling, Fractionation, Chromatography, Mass Spectrometry, Quantitation Assay, Western Blot