ltq orbitrap velos mass spectrometer  (Thermo Fisher)


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    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 2339 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Differential proteomic analysis of synovial fluid from rheumatoid arthritis and osteoarthritis patients"

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

    Journal: Clinical proteomics

    doi: 10.1186/1559-0275-11-1

    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.
    Figure Legend 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.

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

    2) Product Images from "LC–MS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography"

    Article Title: LC–MS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography

    Journal: Journal of Proteome Research

    doi: 10.1021/pr500570m

    Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant differences in expressions between DF versus EAC with p value
    Figure Legend Snippet: Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant differences in expressions between DF versus EAC with p value

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Spectral counts quantitation of LAC-enriched glycoproteins (A) and HC-enriched glycoproteins (B) by LC–ESI–MS/MS that were evaluated as significant differentiated ones between DF and HGD and between DF and EAC with p value
    Figure Legend Snippet: Spectral counts quantitation of LAC-enriched glycoproteins (A) and HC-enriched glycoproteins (B) by LC–ESI–MS/MS that were evaluated as significant differentiated ones between DF and HGD and between DF and EAC with p value

    Techniques Used: Quantitation Assay, Mass Spectrometry

    Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant difference between DF versus EAC and DF versus HGD with p value
    Figure Legend Snippet: Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant difference between DF versus EAC and DF versus HGD with p value

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Principal component analysis (PCA) scores plot for LC–ESI–MS/MS results of LAC- (A) and HC-enriched (B) samples from human blood serum associated with DF subjects ( N = 15, green), HGD ( N = 12 for LAC enrichment and N = 11 for HC enrichment, blue), and EAC ( N = 15, red).
    Figure Legend Snippet: Principal component analysis (PCA) scores plot for LC–ESI–MS/MS results of LAC- (A) and HC-enriched (B) samples from human blood serum associated with DF subjects ( N = 15, green), HGD ( N = 12 for LAC enrichment and N = 11 for HC enrichment, blue), and EAC ( N = 15, red).

    Techniques Used: Mass Spectrometry

    Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant differences in expressions between DF versus HGD with p value
    Figure Legend Snippet: Box and dot plots of normalized peak areas of glycopeptides determined by MRM LC–MS/MS analyses of LAC-enriched samples and glycosylation sites of HC-enriched samples. These are peptides and glycopeptides that have demonstrated a statistically significant differences in expressions between DF versus HGD with p value

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    3) Product Images from "Deciphering preferential interactions within supramolecular protein complexes: the proteasome case"

    Article Title: Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20145497

    Protein correlation profiling (PCP) analysis of glycerol density gradient-separated proteasome complexes PCP-MS strategy to identify proteins interacting with specific proteasome subtypes. U937 cells were cross-linked with formaldehyde and lysed, and proteins were concentrated and ultrafiltrated on a 100 kDa cutoff device. Protein complexes were then separated on a 15–40% glycerol gradient. Each fraction of the gradient was analyzed by nano-LC-MS/MS. Protein quantification was performed using the mean XIC of the three most intense validated peptides for each protein, after internal standard calibration using a mix of 8 isotopically labeled peptides. The PCP analysis was performed as described in the Materials and Methods section. PCP analysis of the 19S regulatory complex. Protein abundance profiles of 16 proteins of the 19S RP (Rpt1–6, Rpn1–3, Rpn5, 7–9, 11–13, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ 2 values (representing the Euclidian distance between the abundance profile of each protein and the reference profile) of the experimental replicate 2 as a function of the χ 2 values of the experimental replicate 1 (middle left panel). The median profile of the 19S complex subunits was used as the reference profile for the calculation of the χ 2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and blue dots represent 19S subunits (right panel). PCP analysis of proteasome 20S complex. Protein abundance profiles of 17 proteins of the 20S CP (α1–α7, β1–β7, β1i, β2i, β5i, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ 2 values of the experimental replicate 2 as a function of the χ 2 values of the experimental replicate 1 (middle left panel). The median profile of the 20S complex subunits was used as the reference profile for the calculation of the χ 2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and red dots represent 20S subunits.
    Figure Legend Snippet: Protein correlation profiling (PCP) analysis of glycerol density gradient-separated proteasome complexes PCP-MS strategy to identify proteins interacting with specific proteasome subtypes. U937 cells were cross-linked with formaldehyde and lysed, and proteins were concentrated and ultrafiltrated on a 100 kDa cutoff device. Protein complexes were then separated on a 15–40% glycerol gradient. Each fraction of the gradient was analyzed by nano-LC-MS/MS. Protein quantification was performed using the mean XIC of the three most intense validated peptides for each protein, after internal standard calibration using a mix of 8 isotopically labeled peptides. The PCP analysis was performed as described in the Materials and Methods section. PCP analysis of the 19S regulatory complex. Protein abundance profiles of 16 proteins of the 19S RP (Rpt1–6, Rpn1–3, Rpn5, 7–9, 11–13, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ 2 values (representing the Euclidian distance between the abundance profile of each protein and the reference profile) of the experimental replicate 2 as a function of the χ 2 values of the experimental replicate 1 (middle left panel). The median profile of the 19S complex subunits was used as the reference profile for the calculation of the χ 2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and blue dots represent 19S subunits (right panel). PCP analysis of proteasome 20S complex. Protein abundance profiles of 17 proteins of the 20S CP (α1–α7, β1–β7, β1i, β2i, β5i, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ 2 values of the experimental replicate 2 as a function of the χ 2 values of the experimental replicate 1 (middle left panel). The median profile of the 20S complex subunits was used as the reference profile for the calculation of the χ 2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and red dots represent 20S subunits.

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

    Changes in the expression of 20S proteasome catalytic subunits modulate 20S-associated regulators The two HEK EBNA cell lines express only standard proteasome or immunoproteasome subunits. Western blots against the immuno- (β1i, β2i, β5i) and standard (β1, β2, β5) catalytic subunits of the 20S proteasome. Calnexin is used as a loading control. Black lines delineate the boundary between vertically sliced images that juxtapose lanes that were non-adjacent in the gel. Importantly, the bands were assembled from the same blot. Relative normalized abundance indexes of proteasome regulators in HEK EBNA cells containing only immunoproteasome compared to HEK EBNA cells containing only standard proteasome. The normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions ( n = 4). Kinetics of IFN-γ treatment on HeLa cells. HeLa cells were stimulated for 0, 24, 48, or 72 h with IFN-γ. Western blots were performed on total cell lysates with antibodies against the β2i, α2, and α5 subunits. IRF-1 was used to control IFN-γ treatment efficiency, and GAPDH was used as a loading control. For each time point of the IFN-γ treatment, proteasome complexes were purified and analyzed by LC-MS/MS. Proteasome complexes dynamics was measured by label-free quantitative proteomics. The normalized abundance index of each protein or protein complex obtained at each time point was compared to the one obtained at the 0 h time point to obtain a regulator relative normalized PAI ( n = 3). Data information: * P
    Figure Legend Snippet: Changes in the expression of 20S proteasome catalytic subunits modulate 20S-associated regulators The two HEK EBNA cell lines express only standard proteasome or immunoproteasome subunits. Western blots against the immuno- (β1i, β2i, β5i) and standard (β1, β2, β5) catalytic subunits of the 20S proteasome. Calnexin is used as a loading control. Black lines delineate the boundary between vertically sliced images that juxtapose lanes that were non-adjacent in the gel. Importantly, the bands were assembled from the same blot. Relative normalized abundance indexes of proteasome regulators in HEK EBNA cells containing only immunoproteasome compared to HEK EBNA cells containing only standard proteasome. The normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions ( n = 4). Kinetics of IFN-γ treatment on HeLa cells. HeLa cells were stimulated for 0, 24, 48, or 72 h with IFN-γ. Western blots were performed on total cell lysates with antibodies against the β2i, α2, and α5 subunits. IRF-1 was used to control IFN-γ treatment efficiency, and GAPDH was used as a loading control. For each time point of the IFN-γ treatment, proteasome complexes were purified and analyzed by LC-MS/MS. Proteasome complexes dynamics was measured by label-free quantitative proteomics. The normalized abundance index of each protein or protein complex obtained at each time point was compared to the one obtained at the 0 h time point to obtain a regulator relative normalized PAI ( n = 3). Data information: * P

    Techniques Used: Expressing, Western Blot, Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Protein abundance correlation of affinity-purified complexes analyzed by mass spectrometry analysis applied to the proteasome complexes and their interacting proteins Heat-map representing the correlations (expressed as the R 2 ) between the abundances of 73 known proteasome-interacting proteins (PIPs) and the abundances of 8 reference proteins or protein complexes, PA28γ, β2i (representing the iP20S), PA28αβ, ncP20S (median of α1–α7, β3, β4, β6, and β7 profiles), 19S (median of Rpt1–6, Rpn1–3, 5–14 profiles), PI31, β5 (representing the sP20S), and PA200. For protein complexes, the median PAI of their subunits in each of the 24 AP-MS experiments was used: α1–α7, β3, β4, β6, and β7 subunits for the ncP20S, Rpt1–6, Rpn1–3, 5–14 for the 19S RP, and PA28α and PA28β subunits for the PA28αβ RP. The R 2 values were hierarchically clustered. Three distinct clusters of composition detailed hereafter could be obtained. Cluster 1 (from top to bottom): Rpt3, Rpn13, α2, Rpn7, USP14, hHR23B, α1, β6, β3, α4, α7, Rpn6, Rpn3, Rpt4, Rpn10, Rpn5, Rpt5, Rpn1, Rpn11, Rpt1, Rpn9, Rpn2, Rpn8, α3, α6, β4, Rpt6, PDC6, Rpn12, APEH, Ubiquilin-1, α5, β7, CCT7, CCT4, CCT2, CCT3, CCT5, CCT6A, DNAJA1, HSP90AB1, HSP90AA1, PNP, Rpt2. Cluster 2 (from top to bottom): 14-3-3ζ/δ, CAND1, GSR, UBE3C, β2, ATP5A1, ATP5B, β1, PA200, β5, FBXO7, UCHL5, TXNL1, ECM29, PI31. Cluster 3 (from top to bottom): PA28β, PITH1, β2i, PA28α, PA28γ, β5i, β1i. Principal component analysis (PCA) of the abundances of 73 known PIPs. The circles represent the main clusters observed (iP20S, ncP20S/19S, sP20S and the 20S assembly chaperones). Plot of the R 2 values between the iP20S or the sP20S and 193 protein correlating ( R 2 > 0.8) with the iP20S, the sP20S, or the ncP20S.
    Figure Legend Snippet: Protein abundance correlation of affinity-purified complexes analyzed by mass spectrometry analysis applied to the proteasome complexes and their interacting proteins Heat-map representing the correlations (expressed as the R 2 ) between the abundances of 73 known proteasome-interacting proteins (PIPs) and the abundances of 8 reference proteins or protein complexes, PA28γ, β2i (representing the iP20S), PA28αβ, ncP20S (median of α1–α7, β3, β4, β6, and β7 profiles), 19S (median of Rpt1–6, Rpn1–3, 5–14 profiles), PI31, β5 (representing the sP20S), and PA200. For protein complexes, the median PAI of their subunits in each of the 24 AP-MS experiments was used: α1–α7, β3, β4, β6, and β7 subunits for the ncP20S, Rpt1–6, Rpn1–3, 5–14 for the 19S RP, and PA28α and PA28β subunits for the PA28αβ RP. The R 2 values were hierarchically clustered. Three distinct clusters of composition detailed hereafter could be obtained. Cluster 1 (from top to bottom): Rpt3, Rpn13, α2, Rpn7, USP14, hHR23B, α1, β6, β3, α4, α7, Rpn6, Rpn3, Rpt4, Rpn10, Rpn5, Rpt5, Rpn1, Rpn11, Rpt1, Rpn9, Rpn2, Rpn8, α3, α6, β4, Rpt6, PDC6, Rpn12, APEH, Ubiquilin-1, α5, β7, CCT7, CCT4, CCT2, CCT3, CCT5, CCT6A, DNAJA1, HSP90AB1, HSP90AA1, PNP, Rpt2. Cluster 2 (from top to bottom): 14-3-3ζ/δ, CAND1, GSR, UBE3C, β2, ATP5A1, ATP5B, β1, PA200, β5, FBXO7, UCHL5, TXNL1, ECM29, PI31. Cluster 3 (from top to bottom): PA28β, PITH1, β2i, PA28α, PA28γ, β5i, β1i. Principal component analysis (PCA) of the abundances of 73 known PIPs. The circles represent the main clusters observed (iP20S, ncP20S/19S, sP20S and the 20S assembly chaperones). Plot of the R 2 values between the iP20S or the sP20S and 193 protein correlating ( R 2 > 0.8) with the iP20S, the sP20S, or the ncP20S.

    Techniques Used: Affinity Purification, Mass Spectrometry

    4) Product Images from "Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts"

    Article Title: Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts

    Journal: Clinical Proteomics

    doi: 10.1186/s12014-018-9197-x

    Phosphotyrosine profiling of cancer epithelial cells and interacting CAFs. a , b Density scatter plot of log 2 transformed phosphopeptide intensity ratios (82T-co-cultured vs. 82T (A) and MDA-MB-231-co-cultured vs. MDA-MB-231) from two SILAC biological experiments. c Pie chart showing the composition of pTyr and pSer/Thr peptides identified in the phosphoproteomic analysis. d Venn diagram showing overlap of phosphopeptides identified in MDA-MB-231 and 82T cells. e , f Gene ontology analysis of phosphoproteins in cancer epithelium and CAFs. e Cellular component; f molecular functions
    Figure Legend Snippet: Phosphotyrosine profiling of cancer epithelial cells and interacting CAFs. a , b Density scatter plot of log 2 transformed phosphopeptide intensity ratios (82T-co-cultured vs. 82T (A) and MDA-MB-231-co-cultured vs. MDA-MB-231) from two SILAC biological experiments. c Pie chart showing the composition of pTyr and pSer/Thr peptides identified in the phosphoproteomic analysis. d Venn diagram showing overlap of phosphopeptides identified in MDA-MB-231 and 82T cells. e , f Gene ontology analysis of phosphoproteins in cancer epithelium and CAFs. e Cellular component; f molecular functions

    Techniques Used: Transformation Assay, Cell Culture, Multiple Displacement Amplification

    5) Product Images from "Tissue Specific Dysregulated Protein Subnetworks in Type 2 Diabetic Bladder Urothelium and Detrusor Muscle *"

    Article Title: Tissue Specific Dysregulated Protein Subnetworks in Type 2 Diabetic Bladder Urothelium and Detrusor Muscle *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M114.041863

    Novel network showing importance of ERK1/2 regulation in the urothelium. Red/pink nodes are proteins that are up-regulated in diabetes, whereas dark/light green nodes are molecules that are down-regulated. Enzymes are represented by diamonds, complexes
    Figure Legend Snippet: Novel network showing importance of ERK1/2 regulation in the urothelium. Red/pink nodes are proteins that are up-regulated in diabetes, whereas dark/light green nodes are molecules that are down-regulated. Enzymes are represented by diamonds, complexes

    Techniques Used:

    The top 10 most significant canonical pathways in urothelium. Blue bars represent the −log( p value) for a given pathway, the larger the bar, the more significant the pathway. The moving orange line with squares is representative of the ratio of
    Figure Legend Snippet: The top 10 most significant canonical pathways in urothelium. Blue bars represent the −log( p value) for a given pathway, the larger the bar, the more significant the pathway. The moving orange line with squares is representative of the ratio of

    Techniques Used:

    A label-free proteomics approach was applied to monitor changes in the soluble proteomes of urothelium and detrusor muscle of diabetic mice (TallyHo) compared with their age-matched controls (SWR/J). Four biological replicates for each tissue type and
    Figure Legend Snippet: A label-free proteomics approach was applied to monitor changes in the soluble proteomes of urothelium and detrusor muscle of diabetic mice (TallyHo) compared with their age-matched controls (SWR/J). Four biological replicates for each tissue type and

    Techniques Used: Mouse Assay

    Differentially Expressed Proteins in Urothelium
    Figure Legend Snippet: Differentially Expressed Proteins in Urothelium

    Techniques Used:

    Validated targets in urothelium by Western blotting.
    Figure Legend Snippet: Validated targets in urothelium by Western blotting.

    Techniques Used: Western Blot

    6) Product Images from "Accurate Label-Free Protein Quantitation with High- and Low-Resolution Mass Spectrometers"

    Article Title: Accurate Label-Free Protein Quantitation with High- and Low-Resolution Mass Spectrometers

    Journal: Journal of proteome research

    doi: 10.1021/pr401017h

    Comparison of label-free quantitation of UPS2 proteins to known mole fraction. UPS2 human protein standards were spiked into an E. coli extract. Identical samples were run on Orbitrap, Velos, and LTQ mass spectrometers. Points from dilutions at 10 −4
    Figure Legend Snippet: Comparison of label-free quantitation of UPS2 proteins to known mole fraction. UPS2 human protein standards were spiked into an E. coli extract. Identical samples were run on Orbitrap, Velos, and LTQ mass spectrometers. Points from dilutions at 10 −4

    Techniques Used: Quantitation Assay

    7) Product Images from "Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates *"

    Article Title: Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M115.055384

    The majority of uncharacterized false positive methyl-PSMs in S. cerevisiae cell lysate samples are predicted by decoy database searches in ETD- and CID-derived datasets but not in HCD-derived datasets. A , proportions of total non-redundant false positive methyl-PSMs that can be explained by equal or higher scoring PSMs with alternative sites of arginine or lysine methylation, cysteinyl- S -β-propionamide, or methylated glutamic or aspartic acid residues, as shown for combined SDS-PAGE (Coomassie stained and unstained)- and HILIC-derived datasets. Remaining false positive methyl-PSMs are considered uncharacterized. Proportions of uncharacterized false positive methyl-PSMs predicted by the target-decoy approach (using separate methyl-PSM FDR estimates) are shown. B , average amino acid compositions of decoy lysine and arginine methyl-PSMs relative to respective high confidence unmethylated lysine- and arginine-containing PSMs (above), and the difference between the average amino acid compositions of decoy methyl-PSMs and uncharacterized false positive PSMs from target database searches (below). For each amino acid, numbers of mass differentials between single amino acids that match to mass differentials associated with mono-, di-, or tri-methylation (14.0157, 28.0314, and 42.0470 Da, respectively) are listed. Amino acids with no such mass differentials are under-represented in decoy methyl-PSMs ( light gray boxes ). ETD data were from an LTQ Orbitrap Velos Pro ETD instrument; CID data were from an LTQ Orbitrap Velos Pro instrument; HCD data were from a Q Exactive Plus instrument. All data are from PSMs of Mascot Except value of
    Figure Legend Snippet: The majority of uncharacterized false positive methyl-PSMs in S. cerevisiae cell lysate samples are predicted by decoy database searches in ETD- and CID-derived datasets but not in HCD-derived datasets. A , proportions of total non-redundant false positive methyl-PSMs that can be explained by equal or higher scoring PSMs with alternative sites of arginine or lysine methylation, cysteinyl- S -β-propionamide, or methylated glutamic or aspartic acid residues, as shown for combined SDS-PAGE (Coomassie stained and unstained)- and HILIC-derived datasets. Remaining false positive methyl-PSMs are considered uncharacterized. Proportions of uncharacterized false positive methyl-PSMs predicted by the target-decoy approach (using separate methyl-PSM FDR estimates) are shown. B , average amino acid compositions of decoy lysine and arginine methyl-PSMs relative to respective high confidence unmethylated lysine- and arginine-containing PSMs (above), and the difference between the average amino acid compositions of decoy methyl-PSMs and uncharacterized false positive PSMs from target database searches (below). For each amino acid, numbers of mass differentials between single amino acids that match to mass differentials associated with mono-, di-, or tri-methylation (14.0157, 28.0314, and 42.0470 Da, respectively) are listed. Amino acids with no such mass differentials are under-represented in decoy methyl-PSMs ( light gray boxes ). ETD data were from an LTQ Orbitrap Velos Pro ETD instrument; CID data were from an LTQ Orbitrap Velos Pro instrument; HCD data were from a Q Exactive Plus instrument. All data are from PSMs of Mascot Except value of

    Techniques Used: Derivative Assay, Methylation, SDS Page, Staining, Hydrophilic Interaction Liquid Chromatography

    8) Product Images from "Differential proteomic analysis of synovial fluid from rheumatoid arthritis and osteoarthritis patients"

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

    Journal: Clinical proteomics

    doi: 10.1186/1559-0275-11-1

    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.
    Figure Legend 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.

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

    9) Product Images from "Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts"

    Article Title: Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts

    Journal: Clinical Proteomics

    doi: 10.1186/s12014-018-9197-x

    Phosphotyrosine profiling of cancer epithelial cells and interacting CAFs. a , b Density scatter plot of log 2 transformed phosphopeptide intensity ratios (82T-co-cultured vs. 82T (A) and MDA-MB-231-co-cultured vs. MDA-MB-231) from two SILAC biological experiments. c Pie chart showing the composition of pTyr and pSer/Thr peptides identified in the phosphoproteomic analysis. d Venn diagram showing overlap of phosphopeptides identified in MDA-MB-231 and 82T cells. e , f Gene ontology analysis of phosphoproteins in cancer epithelium and CAFs. e Cellular component; f molecular functions
    Figure Legend Snippet: Phosphotyrosine profiling of cancer epithelial cells and interacting CAFs. a , b Density scatter plot of log 2 transformed phosphopeptide intensity ratios (82T-co-cultured vs. 82T (A) and MDA-MB-231-co-cultured vs. MDA-MB-231) from two SILAC biological experiments. c Pie chart showing the composition of pTyr and pSer/Thr peptides identified in the phosphoproteomic analysis. d Venn diagram showing overlap of phosphopeptides identified in MDA-MB-231 and 82T cells. e , f Gene ontology analysis of phosphoproteins in cancer epithelium and CAFs. e Cellular component; f molecular functions

    Techniques Used: Transformation Assay, Cell Culture, Multiple Displacement Amplification

    10) Product Images from "A Pan-specific Antibody for Direct Detection of Protein Histidine Phosphorylation"

    Article Title: A Pan-specific Antibody for Direct Detection of Protein Histidine Phosphorylation

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.1259

    pHis levels in PpsA are sensitive to nitrogen availability and are regulated by α-KG a) Western blotting of NCM 3722 cells grown on minimal media containing glucose or glycerol as the carbon source and arginine as the nitrogen source. α-pHis signals were sensitive to hydroxylamine (HA) treatment of the lysates and nitrogen upshift in the growth media (NH 4 Cl). As a loading control, the membranes were imaged with colloidal gold stain ( Supplementary Fig. 21 ). See Supplementary Fig. 22 for full Western blot. b) MS/MS of an endogenous PpsA tryptic pHis peptide identified from fractionated glucose-fed E. coli lysate ( Supplementary Fig. 16 ). The gel band at 85 kDa was analyzed by high-resolution nano-UPLC-MS after trypsin digestion. The spectrum indicates pHis at the canonical His421 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (CAM = S-carboxyamidomethyl) (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). c) Model for regulation of PpsA catalytic cycle. Intracellular levels of α-KG can be significantly increased by nitrogen limitation. Inhibition of the PpsA dephosphorylation by the increased α-KG will lead to the buildup of phosphorylated enzyme. d) Dephosphorylation assay of autophosphorylated PpsA. The dephosphorylation was inhibited by α-KG, but not by glutamate (n = 3, mean ± s.d.).
    Figure Legend Snippet: pHis levels in PpsA are sensitive to nitrogen availability and are regulated by α-KG a) Western blotting of NCM 3722 cells grown on minimal media containing glucose or glycerol as the carbon source and arginine as the nitrogen source. α-pHis signals were sensitive to hydroxylamine (HA) treatment of the lysates and nitrogen upshift in the growth media (NH 4 Cl). As a loading control, the membranes were imaged with colloidal gold stain ( Supplementary Fig. 21 ). See Supplementary Fig. 22 for full Western blot. b) MS/MS of an endogenous PpsA tryptic pHis peptide identified from fractionated glucose-fed E. coli lysate ( Supplementary Fig. 16 ). The gel band at 85 kDa was analyzed by high-resolution nano-UPLC-MS after trypsin digestion. The spectrum indicates pHis at the canonical His421 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (CAM = S-carboxyamidomethyl) (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). c) Model for regulation of PpsA catalytic cycle. Intracellular levels of α-KG can be significantly increased by nitrogen limitation. Inhibition of the PpsA dephosphorylation by the increased α-KG will lead to the buildup of phosphorylated enzyme. d) Dephosphorylation assay of autophosphorylated PpsA. The dephosphorylation was inhibited by α-KG, but not by glutamate (n = 3, mean ± s.d.).

    Techniques Used: Western Blot, Staining, Mass Spectrometry, Sequencing, Chick Chorioallantoic Membrane Assay, Mass Measurement, Inhibition, De-Phosphorylation Assay

    Analysis of histidine phosphorylation on PtsI a) Western blots of E. coli lysates expressing His 6 -tagged PtsI. Left: α-pHis blot of crude lysates and those treated with hydroxylamine (HA) or phosphohistidine phosphatase (PH). Right: Crude lysates were purified over Ni-NTA beads and the indicated fractions probed with the α-pHis antibody. As loading controls, the membranes were stripped and re-blotted with an α-His-tag antibody. See Supplementary Fig. 20 for full Western blots. b) Overexpressed PtsI was digested with trypsin and analyzed by high-resolution nano-UPLC-MS. Shown is the MS/MS spectrum from the tryptic peptide ion bearing pHis at the canonical His-189 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). For comparison, the MS/MS spectrum of a synthetic version of the pHis-bearing peptide is shown in mirror image below. c) A dot blot assay was developed to measure the kinetics of autophosphorylation of PtsI by PEP. A plot of the reaction velocity as a function of PEP concentration was used to determine an apparent K m value of 135 ± 30 μM (n = 3, mean ± s.d.).
    Figure Legend Snippet: Analysis of histidine phosphorylation on PtsI a) Western blots of E. coli lysates expressing His 6 -tagged PtsI. Left: α-pHis blot of crude lysates and those treated with hydroxylamine (HA) or phosphohistidine phosphatase (PH). Right: Crude lysates were purified over Ni-NTA beads and the indicated fractions probed with the α-pHis antibody. As loading controls, the membranes were stripped and re-blotted with an α-His-tag antibody. See Supplementary Fig. 20 for full Western blots. b) Overexpressed PtsI was digested with trypsin and analyzed by high-resolution nano-UPLC-MS. Shown is the MS/MS spectrum from the tryptic peptide ion bearing pHis at the canonical His-189 site, with the matched b- and y- ions indicated in the spectrum and in the sequence flag diagram above (inset: MS spectrum of the precursor ion species, including its accurate mass measurement). For comparison, the MS/MS spectrum of a synthetic version of the pHis-bearing peptide is shown in mirror image below. c) A dot blot assay was developed to measure the kinetics of autophosphorylation of PtsI by PEP. A plot of the reaction velocity as a function of PEP concentration was used to determine an apparent K m value of 135 ± 30 μM (n = 3, mean ± s.d.).

    Techniques Used: Western Blot, Expressing, Purification, Mass Spectrometry, Sequencing, Mass Measurement, Dot Blot, Concentration Assay

    11) Product Images from "Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis"

    Article Title: Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

    Journal: Journal of the American Society for Mass Spectrometry

    doi: 10.1007/s13361-013-0658-1

    Summary of robotic workflow. (a) Starting position: DBS is mounted on the microtitre plate. One well contains extraction solvent and a second well contains trypsin solution; (b) 7 μL of 50 mMol NH 4 HCO 3 is aspirated from solvent well; (c) 6 μL is dispensed onto DBS surface. Liquid microjunction is maintained between the pipette tip and the DBS surface (for 4 s) allowing intact proteins to dissolve into solvent; (d) solution of intact proteins (5 μL) is re-aspirated and dispensed into clean sample well; (e) 4.5 μL of 0.1 μg/μL trypsin solution is aspirated from trypsin well; (f) trypsin solution is added to sample well; (g) sample is incubated at 40 °C for 1 h. Enzyme digests intact proteins into peptides; (h) and (i) as solvent begins to evaporate from sample well, additional solvent (7.5 μL) is aspirated from solvent well and added to sample well [ (h) and (i) are performed at 30 min and 1 h]. (j) Proteins are digested into peptides after 1 h. (k) Plate is transferred to HPLC autosampler and peptides are analyzed by LC MS/MS
    Figure Legend Snippet: Summary of robotic workflow. (a) Starting position: DBS is mounted on the microtitre plate. One well contains extraction solvent and a second well contains trypsin solution; (b) 7 μL of 50 mMol NH 4 HCO 3 is aspirated from solvent well; (c) 6 μL is dispensed onto DBS surface. Liquid microjunction is maintained between the pipette tip and the DBS surface (for 4 s) allowing intact proteins to dissolve into solvent; (d) solution of intact proteins (5 μL) is re-aspirated and dispensed into clean sample well; (e) 4.5 μL of 0.1 μg/μL trypsin solution is aspirated from trypsin well; (f) trypsin solution is added to sample well; (g) sample is incubated at 40 °C for 1 h. Enzyme digests intact proteins into peptides; (h) and (i) as solvent begins to evaporate from sample well, additional solvent (7.5 μL) is aspirated from solvent well and added to sample well [ (h) and (i) are performed at 30 min and 1 h]. (j) Proteins are digested into peptides after 1 h. (k) Plate is transferred to HPLC autosampler and peptides are analyzed by LC MS/MS

    Techniques Used: Transferring, Incubation, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    12) Product Images from "Noncovalently Associated Peptides Observed during Liquid Chromatography-Mass Spectrometry and Their Effect on Cross-Link Analyses"

    Article Title: Noncovalently Associated Peptides Observed during Liquid Chromatography-Mass Spectrometry and Their Effect on Cross-Link Analyses

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.8b04037

    Quality control after cross-link identification at a 5% link FDR. (a) Results from cross-linking HSA with sulfo-SDA acquired on an Q Exactive and an LTQ Orbitrap Velos mass spectrometer. The line at 25 Å indicates the distance cutoff for links classified as long distance. The inlet shows the fraction of long-distance links (LDL) in each data set. (b) Score comparison between within-distance linkzs and LDL. LDL showed no significant (ns) deviation from the within-distance links (two-sided Mann–Whitney-U-test at α = 0.05).
    Figure Legend Snippet: Quality control after cross-link identification at a 5% link FDR. (a) Results from cross-linking HSA with sulfo-SDA acquired on an Q Exactive and an LTQ Orbitrap Velos mass spectrometer. The line at 25 Å indicates the distance cutoff for links classified as long distance. The inlet shows the fraction of long-distance links (LDL) in each data set. (b) Score comparison between within-distance linkzs and LDL. LDL showed no significant (ns) deviation from the within-distance links (two-sided Mann–Whitney-U-test at α = 0.05).

    Techniques Used: Mass Spectrometry, MANN-WHITNEY

    13) Product Images from "Quantitative Proteome Analysis of Temporally Resolved Phagosomes Following Uptake Via Key Phagocytic Receptors *"

    Article Title: Quantitative Proteome Analysis of Temporally Resolved Phagosomes Following Uptake Via Key Phagocytic Receptors *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M114.044594

    Flow diagram of experimental approach for proteomic analysis. Phagosomes were formed by inoculating murine bone marrow-derived macrophages (BMDMs) with polystyrene beads coated with various ligands for 30 min and a subsequent chase for 0, 30 or 150 min. Cells were homogenized and phagosomes isolated through ultracentrifugation in a sucrose gradient. Phagosomal proteins were extracted, digested, and isotopically labeled (light), whereas a mixed pool of phagosome samples that serve as a control was labeled with heavy isotopes. Combined samples were analyzed by LC-MS/MS on an Orbitrap Velos Pro mass spectrometer, data analysis was performed using MaxQuant and Perseus software suites and bioinformatic and statistical analyses were performed using in-house tools.
    Figure Legend Snippet: Flow diagram of experimental approach for proteomic analysis. Phagosomes were formed by inoculating murine bone marrow-derived macrophages (BMDMs) with polystyrene beads coated with various ligands for 30 min and a subsequent chase for 0, 30 or 150 min. Cells were homogenized and phagosomes isolated through ultracentrifugation in a sucrose gradient. Phagosomal proteins were extracted, digested, and isotopically labeled (light), whereas a mixed pool of phagosome samples that serve as a control was labeled with heavy isotopes. Combined samples were analyzed by LC-MS/MS on an Orbitrap Velos Pro mass spectrometer, data analysis was performed using MaxQuant and Perseus software suites and bioinformatic and statistical analyses were performed using in-house tools.

    Techniques Used: Flow Cytometry, Derivative Assay, Isolation, Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Software

    14) Product Images from "ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma"

    Article Title: ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20145450

    Proteomic and genomic workflows Cell lysates from control samples and samples derived from 1 and 3 days after PLX4720 treatment were digested with Lys-C/Trypsin, labeled by triplex dimethyl approach and mixed in 1:1:1 ratios. For protein expression analysis, 200 μg of digested lysate was fractionated by SCX and each fraction was analyzed by LC/MS/MS to determine the relative protein expression levels for every time point compared to the control. For the phosphoproteome, 3 mg of digested lysate was fractionated by SCX and each fraction was enriched for phosphopeptides by Ti 4+ -IMAC prior to LC/MS/MS analysis. Melanoma cells were transduced with a lentiviral kinome library, containing ˜4,000 shRNAs targeting ˜500 kinases. Cells were treated either with DMSO (control) or with BRAFi or ERKi. Genomic DNA was isolated, and hairpins were amplified by PCR. Using deep sequencing, the hairpins that specifically dropped out in the treated sample were identified. In this case, the absence of the blue bar in deep sequencing indicates schematically a synthetic lethal effect of the shRNA and BRAFi/ERKi.
    Figure Legend Snippet: Proteomic and genomic workflows Cell lysates from control samples and samples derived from 1 and 3 days after PLX4720 treatment were digested with Lys-C/Trypsin, labeled by triplex dimethyl approach and mixed in 1:1:1 ratios. For protein expression analysis, 200 μg of digested lysate was fractionated by SCX and each fraction was analyzed by LC/MS/MS to determine the relative protein expression levels for every time point compared to the control. For the phosphoproteome, 3 mg of digested lysate was fractionated by SCX and each fraction was enriched for phosphopeptides by Ti 4+ -IMAC prior to LC/MS/MS analysis. Melanoma cells were transduced with a lentiviral kinome library, containing ˜4,000 shRNAs targeting ˜500 kinases. Cells were treated either with DMSO (control) or with BRAFi or ERKi. Genomic DNA was isolated, and hairpins were amplified by PCR. Using deep sequencing, the hairpins that specifically dropped out in the treated sample were identified. In this case, the absence of the blue bar in deep sequencing indicates schematically a synthetic lethal effect of the shRNA and BRAFi/ERKi.

    Techniques Used: Derivative Assay, Labeling, Expressing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Transduction, Isolation, Amplification, Polymerase Chain Reaction, Sequencing, shRNA

    15) Product Images from "Exploring the Human Plasma Proteome for Humoral Mediators of Remote Ischemic Preconditioning - A Word of Caution"

    Article Title: Exploring the Human Plasma Proteome for Humoral Mediators of Remote Ischemic Preconditioning - A Word of Caution

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0109279

    Analysis workflow. Plasma samples were depleted by a MARS Hu-14 column and subsequently concentrated by 3 kDa ultracentrifugation filters. Next, samples were reduced, cysteine blocked and trypsin digested before iTRAQ labeling. The iTRAQ labeled peptides were fractioned into 60 fractions using a mixed-mode reverse phase anion exchanger. Finally, fractions were analyzed on an LTQ-Orbitrap Velos Pro connected to a Dionex Ultimate NCR-3000RS LC system.
    Figure Legend Snippet: Analysis workflow. Plasma samples were depleted by a MARS Hu-14 column and subsequently concentrated by 3 kDa ultracentrifugation filters. Next, samples were reduced, cysteine blocked and trypsin digested before iTRAQ labeling. The iTRAQ labeled peptides were fractioned into 60 fractions using a mixed-mode reverse phase anion exchanger. Finally, fractions were analyzed on an LTQ-Orbitrap Velos Pro connected to a Dionex Ultimate NCR-3000RS LC system.

    Techniques Used: Labeling

    16) Product Images from "Proteome data of female Anopheles stephensi antennae"

    Article Title: Proteome data of female Anopheles stephensi antennae

    Journal: Data in Brief

    doi: 10.1016/j.dib.2019.103911

    The workflow illustrating the steps involved in proteomic analysis of antennae of female Anopheles stephensi. Proteins were extracted from the antennae and subjected to in-gel trypsin digestion followed by analyses on LTQ-Orbitrap Velos mass spectrometer. Mascot and SEQUEST algorithms were used for database searches.
    Figure Legend Snippet: The workflow illustrating the steps involved in proteomic analysis of antennae of female Anopheles stephensi. Proteins were extracted from the antennae and subjected to in-gel trypsin digestion followed by analyses on LTQ-Orbitrap Velos mass spectrometer. Mascot and SEQUEST algorithms were used for database searches.

    Techniques Used: Mass Spectrometry

    17) Product Images from "Identifying Kinase Substrates via a Heavy ATP Kinase Assay and Quantitative Mass Spectrometry"

    Article Title: Identifying Kinase Substrates via a Heavy ATP Kinase Assay and Quantitative Mass Spectrometry

    Journal: Scientific Reports

    doi: 10.1038/srep28107

    Spectral features of HAKA-MS. An example of an MS 2 spectrum corresponding to a tryptic peptide derived from SRC8 containing a reported ABL1 phosphorylation site. The assigned fragment ions provide peptide-specific sequence information that is used to computationally match the data to the corresponding peptide in a protein database. Certain fragment ions ( i.e. , y 8 and b 7 ) unambiguously assign the phosphate group (+84 Da) to Y421 and not to S417, S418 or S426. ( a ) Magnification of the TMT reporter ion region to illustrate the relative abundance of the individual multiplexed samples for this specific phosphotyrosine-containing peptide. TMT 6-plex ion intensities were used to generate individual relative ratios or to plot reaction progress kinetics. ( b ) pY-immonium ions for ‘light’ ( m/z 216.04) and ‘heavy’ ( m/z 220.04) versions of the phosphopeptide. The existence and relative abundances of the immonium ions provide unequivocal evidence of tyrosine residue phosphorylation and quality control of the respective spectrum. ( c ) MS 1 precursor ion of the phosphotyrosine-containing peptide with both the ‘light’ ( m/z 683.6848) and ‘heavy’ ( m/z 685.0192) isotopologues. Both isotopologues (blue and red peaks) display a typical 12 C/ 13 C-isotopic peak distribution (1 a.m.u. separation) but with a slight overlap due to the small ∆ m/z of 1.333 Th. Precursor ion selection of the monoisotopic peak (pink area) was wi thin the isolation window of ±0.4 Th and partially including the ‘light’ isotopologue (fourth 13 C-isotopic peak).
    Figure Legend Snippet: Spectral features of HAKA-MS. An example of an MS 2 spectrum corresponding to a tryptic peptide derived from SRC8 containing a reported ABL1 phosphorylation site. The assigned fragment ions provide peptide-specific sequence information that is used to computationally match the data to the corresponding peptide in a protein database. Certain fragment ions ( i.e. , y 8 and b 7 ) unambiguously assign the phosphate group (+84 Da) to Y421 and not to S417, S418 or S426. ( a ) Magnification of the TMT reporter ion region to illustrate the relative abundance of the individual multiplexed samples for this specific phosphotyrosine-containing peptide. TMT 6-plex ion intensities were used to generate individual relative ratios or to plot reaction progress kinetics. ( b ) pY-immonium ions for ‘light’ ( m/z 216.04) and ‘heavy’ ( m/z 220.04) versions of the phosphopeptide. The existence and relative abundances of the immonium ions provide unequivocal evidence of tyrosine residue phosphorylation and quality control of the respective spectrum. ( c ) MS 1 precursor ion of the phosphotyrosine-containing peptide with both the ‘light’ ( m/z 683.6848) and ‘heavy’ ( m/z 685.0192) isotopologues. Both isotopologues (blue and red peaks) display a typical 12 C/ 13 C-isotopic peak distribution (1 a.m.u. separation) but with a slight overlap due to the small ∆ m/z of 1.333 Th. Precursor ion selection of the monoisotopic peak (pink area) was wi thin the isolation window of ±0.4 Th and partially including the ‘light’ isotopologue (fourth 13 C-isotopic peak).

    Techniques Used: Mass Spectrometry, Derivative Assay, Sequencing, Selection, Isolation

    Schematic workflow of HAKA-MS. Cytosolic fractions were treated with the pan-kinase inhibitor FSBA to irreversibly abolish cellular kinase activity. Simultaneous exchange to the kinase assay buffer; and removal of excess FSBA and endogenous ATP was achieved by ultrafiltration. In parallel, constitutively-active ABL1-PP and inactive ABL1-Kin − were immunopurified via protein G sepharose beads. Prior to the kinase assay, treated lysate and washed kinase-bead fractions were equally divided, mixed and the kinase reaction initiated with ‘heavy’ 18 O-labelled ATP. To obtain the phosphorylation kinetic series, pairs of active/inactive kinase reactions were quenched at defined time points. Following enzymatic digestion, peptides were concentrated with solid-phase extraction and phosphotyrosine-containing peptides enriched via immunoprecipitation. Eluted peptides were chemically-modified with isobaric amine-reactive tandem mass tag (TMT) reagents to multiplex 3 × 2 samples. After a second phosphopeptide enrichment with immobilised metal-affinity chromatography (IMAC), the sample was analysed by LCMS.
    Figure Legend Snippet: Schematic workflow of HAKA-MS. Cytosolic fractions were treated with the pan-kinase inhibitor FSBA to irreversibly abolish cellular kinase activity. Simultaneous exchange to the kinase assay buffer; and removal of excess FSBA and endogenous ATP was achieved by ultrafiltration. In parallel, constitutively-active ABL1-PP and inactive ABL1-Kin − were immunopurified via protein G sepharose beads. Prior to the kinase assay, treated lysate and washed kinase-bead fractions were equally divided, mixed and the kinase reaction initiated with ‘heavy’ 18 O-labelled ATP. To obtain the phosphorylation kinetic series, pairs of active/inactive kinase reactions were quenched at defined time points. Following enzymatic digestion, peptides were concentrated with solid-phase extraction and phosphotyrosine-containing peptides enriched via immunoprecipitation. Eluted peptides were chemically-modified with isobaric amine-reactive tandem mass tag (TMT) reagents to multiplex 3 × 2 samples. After a second phosphopeptide enrichment with immobilised metal-affinity chromatography (IMAC), the sample was analysed by LCMS.

    Techniques Used: Mass Spectrometry, Activity Assay, Kinase Assay, Immunoprecipitation, Modification, Multiplex Assay, Affinity Chromatography, Liquid Chromatography with Mass Spectroscopy

    In vitro substrates identified by HAKA-MS. ( a ) TMT reporter ion intensities of phosphotyrosine-containing peptides were normalised and plotted against the respective kinase assay time point. Connecting lines are shown for active ABL1-PP (orange) or inactive ABL1-Kin − (blue). As ‘heavy’ phosphate groups are absent prior to the addition of 18 O-labelled ATP, the lines were extrapolated through the origin (shaded to indicate hypothetical). A clear separation between the kinetic trend lines for active and inactive ABL1 was apparent. ( b ) All 61 identified in vitro substrates of ABL1 with at least one ‘heavy’ phosphotyrosine residue. Known and validated substrates are shown in green and purple, respectively. Proteins with purple shading contain the preferred ABL1 YxxP consensus motif (p-value ≤0.05; motif-score: 4.63; fold-increase: 3.16; www.motif-x.med.harvard.edu ). Sphere size indicates the number of phosphotyrosines per substrate. ( c ) Comparison of the ABL1-PP/ABL1-Kin − ratio distribution at 90 minutes for HAKA-MS (front) versus the kinase assay performed with normal ATP (back). ‘Light’ and ‘heavy’ pY-containing peptides are labelled in grey and red, respectively. In both cases, the global distribution is bimodal with a shallow, intersecting valley. For the normal ATP kinase assay, the threshold for putative in vitro ABL1 substrates was approximated with mixed Gaussian clustering. The first and second clusters are considered noise and in vitro substrates, respectively. Comparison to HAKA-MS revealed that regardless of the cut-off stringency ( i.e ., pink, ≥ 4 ratio), data from the normal ATP assay can contain many false positives (grey). Conversely, true positives (red) were excluded.
    Figure Legend Snippet: In vitro substrates identified by HAKA-MS. ( a ) TMT reporter ion intensities of phosphotyrosine-containing peptides were normalised and plotted against the respective kinase assay time point. Connecting lines are shown for active ABL1-PP (orange) or inactive ABL1-Kin − (blue). As ‘heavy’ phosphate groups are absent prior to the addition of 18 O-labelled ATP, the lines were extrapolated through the origin (shaded to indicate hypothetical). A clear separation between the kinetic trend lines for active and inactive ABL1 was apparent. ( b ) All 61 identified in vitro substrates of ABL1 with at least one ‘heavy’ phosphotyrosine residue. Known and validated substrates are shown in green and purple, respectively. Proteins with purple shading contain the preferred ABL1 YxxP consensus motif (p-value ≤0.05; motif-score: 4.63; fold-increase: 3.16; www.motif-x.med.harvard.edu ). Sphere size indicates the number of phosphotyrosines per substrate. ( c ) Comparison of the ABL1-PP/ABL1-Kin − ratio distribution at 90 minutes for HAKA-MS (front) versus the kinase assay performed with normal ATP (back). ‘Light’ and ‘heavy’ pY-containing peptides are labelled in grey and red, respectively. In both cases, the global distribution is bimodal with a shallow, intersecting valley. For the normal ATP kinase assay, the threshold for putative in vitro ABL1 substrates was approximated with mixed Gaussian clustering. The first and second clusters are considered noise and in vitro substrates, respectively. Comparison to HAKA-MS revealed that regardless of the cut-off stringency ( i.e ., pink, ≥ 4 ratio), data from the normal ATP assay can contain many false positives (grey). Conversely, true positives (red) were excluded.

    Techniques Used: In Vitro, Mass Spectrometry, Kinase Assay, Significance Assay, ATP Assay

    DDX3X is a novel substrate of ABL1. ( a ) Phosphorylation kinetics for the DDX3X peptide DKDAYSSFGSR. The ‘heavy’ phosphate group is located on Y69. Calculated rate constants ( k ) are plotted for the constitutively-active ABL1-PP and catalytically-inactive ABL1-Kin − . Error bars represent two standard deviations (2σ) from the mean. ( b ) Biochemical validation of DDX3X tyrosine phosphorylation mediated by ABL1. HA-tagged DDX3X was transiently co-transfected in HEK293 cells in the presence of ABL1, ABL1-Kin − , ABL1-PP or the empty vector. DDX3X was immunopurified with HA-beads and the tyrosine phosphorylation state assessed by α-phosphotyrosine (4G10) immunostaining. Total levels of DDX3X-HA, ABL1 and tyrosine-phosphorylated proteins were visualised by immunostaining against α-HA, α-ABL1 and α-phosphotyrosine (4G10), respectively.
    Figure Legend Snippet: DDX3X is a novel substrate of ABL1. ( a ) Phosphorylation kinetics for the DDX3X peptide DKDAYSSFGSR. The ‘heavy’ phosphate group is located on Y69. Calculated rate constants ( k ) are plotted for the constitutively-active ABL1-PP and catalytically-inactive ABL1-Kin − . Error bars represent two standard deviations (2σ) from the mean. ( b ) Biochemical validation of DDX3X tyrosine phosphorylation mediated by ABL1. HA-tagged DDX3X was transiently co-transfected in HEK293 cells in the presence of ABL1, ABL1-Kin − , ABL1-PP or the empty vector. DDX3X was immunopurified with HA-beads and the tyrosine phosphorylation state assessed by α-phosphotyrosine (4G10) immunostaining. Total levels of DDX3X-HA, ABL1 and tyrosine-phosphorylated proteins were visualised by immunostaining against α-HA, α-ABL1 and α-phosphotyrosine (4G10), respectively.

    Techniques Used: Transfection, Plasmid Preparation, Immunostaining

    18) Product Images from "Dysregulation of splicing proteins in head and neck squamous cell carcinoma"

    Article Title: Dysregulation of splicing proteins in head and neck squamous cell carcinoma

    Journal: Cancer Biology & Therapy

    doi: 10.1080/15384047.2016.1139234

    Representative MS and MS/MS spectra of phosphopeptides identified in HNSCC cells (A) serine/arginine-rich splicing factor 2 (SRSF2) (B) SRSF protein kinase 2 (SRPK2) (C) Western blot analysis shows overexpression and hyperphosphorylation of SRPK2 in a
    Figure Legend Snippet: Representative MS and MS/MS spectra of phosphopeptides identified in HNSCC cells (A) serine/arginine-rich splicing factor 2 (SRSF2) (B) SRSF protein kinase 2 (SRPK2) (C) Western blot analysis shows overexpression and hyperphosphorylation of SRPK2 in a

    Techniques Used: Mass Spectrometry, Western Blot, Over Expression

    19) Product Images from "TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics *"

    Article Title: TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M112.017764

    Novel kinases/phosphatases identified in TSLP signaling pathway. A–H , phosphorylation status of tyrosine residues of nonreceptor tyrosine kinases—Btk, Tec, Lyn, Fes, and Hck/Src/Yes1/Fyn/Lck—and nonreceptor tyrosine phosphatases—Ptpn6 and Ptpn18—and PI-3 kinase catalytic subunit were up-regulated as evidenced by MS spectra showing the changes in the relative abundance of phosphopeptides.
    Figure Legend Snippet: Novel kinases/phosphatases identified in TSLP signaling pathway. A–H , phosphorylation status of tyrosine residues of nonreceptor tyrosine kinases—Btk, Tec, Lyn, Fes, and Hck/Src/Yes1/Fyn/Lck—and nonreceptor tyrosine phosphatases—Ptpn6 and Ptpn18—and PI-3 kinase catalytic subunit were up-regulated as evidenced by MS spectra showing the changes in the relative abundance of phosphopeptides.

    Techniques Used: Mass Spectrometry

    A schematic illustration of the SILAC-based quantitative phosphoproteomic approach. Two populations of Ba/F3-IT cells were cultured in “light” or “heavy” medium, separately. The cells grown in heavy medium were stimulated with TSLP for 15 min. After lysis, the samples were mixed, digested with trypsin, desalted on a C 18 column and subjected to lyophilization. One portion of the peptide mixture was incubated with antiphosphotyrosine antibodies for enrichment of tyrosine-phosphorylated peptides. The other part of the peptide mixture was fractionated by SCX and phosphopeptides were enriched on TiO 2 beads. The enriched phosphopeptides were analyzed by LC-MS/MS. The resulting high resolution mass spectra reveal TSLP-induced changes in the phosphorylation status on each site.
    Figure Legend Snippet: A schematic illustration of the SILAC-based quantitative phosphoproteomic approach. Two populations of Ba/F3-IT cells were cultured in “light” or “heavy” medium, separately. The cells grown in heavy medium were stimulated with TSLP for 15 min. After lysis, the samples were mixed, digested with trypsin, desalted on a C 18 column and subjected to lyophilization. One portion of the peptide mixture was incubated with antiphosphotyrosine antibodies for enrichment of tyrosine-phosphorylated peptides. The other part of the peptide mixture was fractionated by SCX and phosphopeptides were enriched on TiO 2 beads. The enriched phosphopeptides were analyzed by LC-MS/MS. The resulting high resolution mass spectra reveal TSLP-induced changes in the phosphorylation status on each site.

    Techniques Used: Cell Culture, Lysis, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Overlap of phosphopeptides and phosphosites identified from antiphosphotyrosine- and TiO 2 -based phosphopeptides enrichment methods. A–C , Overlap of identified ( A ) phosphoserine (pSer)-, ( B ) phosphothreonine (pThr)- and ( C ) phosphotyrosine (pTyr)-containing peptides from antiphosphotyrosine- and TiO2-based phosphopeptides enrichment methods; D–F , Overlap of identified ( D ) pSer ( B ) pThr and ( C ) pTyr sites from anti-phosphotyrosine- and TiO2-based phosphopeptides enrichment methods.
    Figure Legend Snippet: Overlap of phosphopeptides and phosphosites identified from antiphosphotyrosine- and TiO 2 -based phosphopeptides enrichment methods. A–C , Overlap of identified ( A ) phosphoserine (pSer)-, ( B ) phosphothreonine (pThr)- and ( C ) phosphotyrosine (pTyr)-containing peptides from antiphosphotyrosine- and TiO2-based phosphopeptides enrichment methods; D–F , Overlap of identified ( D ) pSer ( B ) pThr and ( C ) pTyr sites from anti-phosphotyrosine- and TiO2-based phosphopeptides enrichment methods.

    Techniques Used:

    20) Product Images from "A proteomic survey of widespread protein aggregation in yeast"

    Article Title: A proteomic survey of widespread protein aggregation in yeast

    Journal: Molecular bioSystems

    doi: 10.1039/c3mb70508k

    Immunopurification of Hsp82-GFP from stationary phase cells co-precipitated a significant number of foci forming proteins A) Among the many interaction partners identified with Hsp82-GFP, Gln1 was reciprocally enriched (combined Z-score > 4.3, fold enrichment > 3.7), as were other foci forming proteins (green bars). B) Of the 50 cytoplasmic proteins that co-purify with Hsp82-GFP, a significant number (21) have been observed to form foci ( p ≤ 10 −3 , Table S3, 811 cytoplasmic proteins as background set), suggesting that a subset of foci-forming proteins are Hsp90 clients in their foci-forming state. C) Partial co-localization of Gln1-GFP with Hsp82-TagRFP in dually fluorescent protein tagged yeast cells grown to stationary phase in synthetic complete medium (Spearman rank correlation r = 0.50 across GFP and TagRFP image fluorescent pixel intensities. Scale bar is 10 μm).
    Figure Legend Snippet: Immunopurification of Hsp82-GFP from stationary phase cells co-precipitated a significant number of foci forming proteins A) Among the many interaction partners identified with Hsp82-GFP, Gln1 was reciprocally enriched (combined Z-score > 4.3, fold enrichment > 3.7), as were other foci forming proteins (green bars). B) Of the 50 cytoplasmic proteins that co-purify with Hsp82-GFP, a significant number (21) have been observed to form foci ( p ≤ 10 −3 , Table S3, 811 cytoplasmic proteins as background set), suggesting that a subset of foci-forming proteins are Hsp90 clients in their foci-forming state. C) Partial co-localization of Gln1-GFP with Hsp82-TagRFP in dually fluorescent protein tagged yeast cells grown to stationary phase in synthetic complete medium (Spearman rank correlation r = 0.50 across GFP and TagRFP image fluorescent pixel intensities. Scale bar is 10 μm).

    Techniques Used: Immu-Puri

    Immunopurification reveals a diversity of foci composition, with a common theme of protein quality control A) Gln1-GFP, while diffuse in log-phase cells, localized predominantly into foci in stationary-phase cells, which persisted in lysate (scale bars are 10μm) and B) could be selectively purified by immunoprecipitation with goat anti-GFP antibodies and visualized by western blot with mouse anti-GFP antibodies. Proteins that co-immunopurified with various GFP-tagged, foci-forming proteins were assayed by shotgun mass spectrometry. Identified proteins are arranged by significance of enrichment relative to the untagged control strain, BY4741, on the X-axis and the fold change of enrichment on the Y-axis for each bait protein tested. C) Immunoprecipitation of Gln1-GFP from stationary phase cells co-immunoprecipitated the cytoplasmic hsp90 class heat shock proteins Hsp82 and Hsc82. D) Ils1-GFP also co-purified with chaperones, but instead with the hsp70 class chaperones Ssb1 and Ssb2. Several other tRNA synthetases also co-purified with Ils1-GFP. E) Cdc19-GFP also co-purified with hsp70 class chaperones as fellow members of glycolysis, notably Pdh1p.
    Figure Legend Snippet: Immunopurification reveals a diversity of foci composition, with a common theme of protein quality control A) Gln1-GFP, while diffuse in log-phase cells, localized predominantly into foci in stationary-phase cells, which persisted in lysate (scale bars are 10μm) and B) could be selectively purified by immunoprecipitation with goat anti-GFP antibodies and visualized by western blot with mouse anti-GFP antibodies. Proteins that co-immunopurified with various GFP-tagged, foci-forming proteins were assayed by shotgun mass spectrometry. Identified proteins are arranged by significance of enrichment relative to the untagged control strain, BY4741, on the X-axis and the fold change of enrichment on the Y-axis for each bait protein tested. C) Immunoprecipitation of Gln1-GFP from stationary phase cells co-immunoprecipitated the cytoplasmic hsp90 class heat shock proteins Hsp82 and Hsc82. D) Ils1-GFP also co-purified with chaperones, but instead with the hsp70 class chaperones Ssb1 and Ssb2. Several other tRNA synthetases also co-purified with Ils1-GFP. E) Cdc19-GFP also co-purified with hsp70 class chaperones as fellow members of glycolysis, notably Pdh1p.

    Techniques Used: Immu-Puri, Purification, Immunoprecipitation, Western Blot, Mass Spectrometry

    Foci are cytoplasmic, insoluble protein assemblies that form independent of a GFP-tag, shown here for the example of glutamine synthetase (Gln1) A) Microscopy of stationary phase yeast cells expressing Gln1-GFP from its native locus or GFP from a plasmid pRS426-GPD-GFPS35T-HIS3. Upon lysis, the Gln1-GFP foci are still visible in the insoluble fraction, in contrast to the GFP only. Scale bars are 10μm. B) Mass spectrometry of the fractionated cell lysate shows the majority of Gln1-GFP is in the insoluble fraction. Similarly untagged, Gln1p is also insoluble whereas the plasmid expressed GFP is soluble. Grey lines indicate the threshold for the 95% confidence interval (|Z| > 1.96).
    Figure Legend Snippet: Foci are cytoplasmic, insoluble protein assemblies that form independent of a GFP-tag, shown here for the example of glutamine synthetase (Gln1) A) Microscopy of stationary phase yeast cells expressing Gln1-GFP from its native locus or GFP from a plasmid pRS426-GPD-GFPS35T-HIS3. Upon lysis, the Gln1-GFP foci are still visible in the insoluble fraction, in contrast to the GFP only. Scale bars are 10μm. B) Mass spectrometry of the fractionated cell lysate shows the majority of Gln1-GFP is in the insoluble fraction. Similarly untagged, Gln1p is also insoluble whereas the plasmid expressed GFP is soluble. Grey lines indicate the threshold for the 95% confidence interval (|Z| > 1.96).

    Techniques Used: Microscopy, Expressing, Plasmid Preparation, Lysis, Mass Spectrometry

    21) Product Images from "Proteomic analysis of human vitreous humor"

    Article Title: Proteomic analysis of human vitreous humor

    Journal: Clinical Proteomics

    doi: 10.1186/1559-0275-11-29

    Experimental design for proteomic characterization of vitreous humor. Pooled vitreous humor samples were depleted of abundant proteins using Agilent’s MARS 14 column followed by in-gel digestion, in-solution digestion and OFFGEL electrophoresis. The samples were analyzed on LTQ-Orbitrap Velos mass spectrometer.
    Figure Legend Snippet: Experimental design for proteomic characterization of vitreous humor. Pooled vitreous humor samples were depleted of abundant proteins using Agilent’s MARS 14 column followed by in-gel digestion, in-solution digestion and OFFGEL electrophoresis. The samples were analyzed on LTQ-Orbitrap Velos mass spectrometer.

    Techniques Used: Electrophoresis, Mass Spectrometry

    22) Product Images from "Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis"

    Article Title: Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.1277

    Inhibition of fatty acyl-AMP loading onto purified Pks13 by TP2 The loading of FadD32-activated FL C 16 on Pks13_WT ( a ), Pks13_F79S ( b ) and MsmPks13 ( c ) was determined by separating the reaction mixtures on SDS-PAGE gels. Activities were determined by measuring in-gel fluorescence (top panels of a , b and c ) and total protein by coomassie blue staining (bottom panels of a , b and c ). SeeBlue Plus2 Pre-stained marker (Invitrogen) was used as molecular weight standard and approximate molecular weights in MOPS running buffer are indicated. The loading of FL C 16 on Pks13_WT ( a ), Pks13_F79S ( b ) and MsmPks13 ( c ) was quantified using ImageQuant 5.2 (GE healthcare) and quantitation from 5-replicates (mean ± SD) each was used to generate inhibition curve ( d ). The full length images corresponding to these figures are shown in Supplementary Fig. 14 .
    Figure Legend Snippet: Inhibition of fatty acyl-AMP loading onto purified Pks13 by TP2 The loading of FadD32-activated FL C 16 on Pks13_WT ( a ), Pks13_F79S ( b ) and MsmPks13 ( c ) was determined by separating the reaction mixtures on SDS-PAGE gels. Activities were determined by measuring in-gel fluorescence (top panels of a , b and c ) and total protein by coomassie blue staining (bottom panels of a , b and c ). SeeBlue Plus2 Pre-stained marker (Invitrogen) was used as molecular weight standard and approximate molecular weights in MOPS running buffer are indicated. The loading of FL C 16 on Pks13_WT ( a ), Pks13_F79S ( b ) and MsmPks13 ( c ) was quantified using ImageQuant 5.2 (GE healthcare) and quantitation from 5-replicates (mean ± SD) each was used to generate inhibition curve ( d ). The full length images corresponding to these figures are shown in Supplementary Fig. 14 .

    Techniques Used: Inhibition, Purification, SDS Page, Fluorescence, Staining, Marker, Molecular Weight, Quantitation Assay

    23) Product Images from "Identification of candidate circulating cisplatin-resistant biomarkers from epithelial ovarian carcinoma cell secretomes"

    Article Title: Identification of candidate circulating cisplatin-resistant biomarkers from epithelial ovarian carcinoma cell secretomes

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2013.687

    Comparison of proteins identified from the current ovarian cancer cell secretome analyses to publicly available databases/data sets, including ( A ) the secreted protein database (SPD) and the Human Proteome Organization (HUPO) plasma proteome; and ( B ) TIF and ascites from ovarian cancer patients.
    Figure Legend Snippet: Comparison of proteins identified from the current ovarian cancer cell secretome analyses to publicly available databases/data sets, including ( A ) the secreted protein database (SPD) and the Human Proteome Organization (HUPO) plasma proteome; and ( B ) TIF and ascites from ovarian cancer patients.

    Techniques Used:

    Immunoblotting verification of selected target secretome proteins. MMP1, MMP10, CTH and COL11A1 were selected for validation by western blotting in EOC secretome.
    Figure Legend Snippet: Immunoblotting verification of selected target secretome proteins. MMP1, MMP10, CTH and COL11A1 were selected for validation by western blotting in EOC secretome.

    Techniques Used: Western Blot

    Venn diagram of 1688 secretome proteins identified with two or more spectral counts from five EOC cell lines.
    Figure Legend Snippet: Venn diagram of 1688 secretome proteins identified with two or more spectral counts from five EOC cell lines.

    Techniques Used:

    24) Product Images from "Laserspray Ionization, a New Method for Protein Analysis Directly from Tissue at Atmospheric Pressure with Ultrahigh Mass Resolution and Electron Transfer Dissociation *"

    Article Title: Laserspray Ionization, a New Method for Protein Analysis Directly from Tissue at Atmospheric Pressure with Ultrahigh Mass Resolution and Electron Transfer Dissociation *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M110.000760

    LSI-MS summed full and inset mass spectra of delipified fresh tissue on plain glass slide spotted with 2,5-DHAP matrix in 50:50 ACN:water showing multiple charged protein ions using Orbitrap Exactive mass spectrometer. Rel. Abund. , relative abundance.
    Figure Legend Snippet: LSI-MS summed full and inset mass spectra of delipified fresh tissue on plain glass slide spotted with 2,5-DHAP matrix in 50:50 ACN:water showing multiple charged protein ions using Orbitrap Exactive mass spectrometer. Rel. Abund. , relative abundance.

    Techniques Used: Mass Spectrometry

    LSI mass spectra from aged mouse brain tissue washed with ethanol and spotted with 2,5-DHAP matrix in 50:50 ACN:water using an Orbitrap Exactive mass spectrometer. A , full mass spectrum with insets showing multiple charged protein and peptide ions. B , limited mass range between m/z 650 and 1000 is displayed with monoisotopic molecular weights of the various multiply charged ions presented. Rel. Abund. , relative abundance.
    Figure Legend Snippet: LSI mass spectra from aged mouse brain tissue washed with ethanol and spotted with 2,5-DHAP matrix in 50:50 ACN:water using an Orbitrap Exactive mass spectrometer. A , full mass spectrum with insets showing multiple charged protein and peptide ions. B , limited mass range between m/z 650 and 1000 is displayed with monoisotopic molecular weights of the various multiply charged ions presented. Rel. Abund. , relative abundance.

    Techniques Used: Mass Spectrometry

    25) Product Images from "Noncovalently Associated Peptides Observed during Liquid Chromatography-Mass Spectrometry and Their Effect on Cross-Link Analyses"

    Article Title: Noncovalently Associated Peptides Observed during Liquid Chromatography-Mass Spectrometry and Their Effect on Cross-Link Analyses

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.8b04037

    Quality control after cross-link identification at a 5% link FDR. (a) Results from cross-linking HSA with sulfo-SDA acquired on an Q Exactive and an LTQ Orbitrap Velos mass spectrometer. The line at 25 Å indicates the distance cutoff for links classified as long distance. The inlet shows the fraction of long-distance links (LDL) in each data set. (b) Score comparison between within-distance linkzs and LDL. LDL showed no significant (ns) deviation from the within-distance links (two-sided Mann–Whitney-U-test at α = 0.05).
    Figure Legend Snippet: Quality control after cross-link identification at a 5% link FDR. (a) Results from cross-linking HSA with sulfo-SDA acquired on an Q Exactive and an LTQ Orbitrap Velos mass spectrometer. The line at 25 Å indicates the distance cutoff for links classified as long distance. The inlet shows the fraction of long-distance links (LDL) in each data set. (b) Score comparison between within-distance linkzs and LDL. LDL showed no significant (ns) deviation from the within-distance links (two-sided Mann–Whitney-U-test at α = 0.05).

    Techniques Used: Mass Spectrometry, MANN-WHITNEY

    26) Product Images from "Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis"

    Article Title: Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

    Journal: Journal of the American Society for Mass Spectrometry

    doi: 10.1007/s13361-013-0658-1

    Summary of robotic workflow. (a) Starting position: DBS is mounted on the microtitre plate. One well contains extraction solvent and a second well contains trypsin solution; (b) 7 μL of 50 mMol NH 4 HCO 3 is aspirated from solvent well; (c) 6 μL is dispensed onto DBS surface. Liquid microjunction is maintained between the pipette tip and the DBS surface (for 4 s) allowing intact proteins to dissolve into solvent; (d) solution of intact proteins (5 μL) is re-aspirated and dispensed into clean sample well; (e) 4.5 μL of 0.1 μg/μL trypsin solution is aspirated from trypsin well; (f) trypsin solution is added to sample well; (g) sample is incubated at 40 °C for 1 h. Enzyme digests intact proteins into peptides; (h) and (i) as solvent begins to evaporate from sample well, additional solvent (7.5 μL) is aspirated from solvent well and added to sample well [ (h) and (i) are performed at 30 min and 1 h]. (j) Proteins are digested into peptides after 1 h. (k) Plate is transferred to HPLC autosampler and peptides are analyzed by LC MS/MS
    Figure Legend Snippet: Summary of robotic workflow. (a) Starting position: DBS is mounted on the microtitre plate. One well contains extraction solvent and a second well contains trypsin solution; (b) 7 μL of 50 mMol NH 4 HCO 3 is aspirated from solvent well; (c) 6 μL is dispensed onto DBS surface. Liquid microjunction is maintained between the pipette tip and the DBS surface (for 4 s) allowing intact proteins to dissolve into solvent; (d) solution of intact proteins (5 μL) is re-aspirated and dispensed into clean sample well; (e) 4.5 μL of 0.1 μg/μL trypsin solution is aspirated from trypsin well; (f) trypsin solution is added to sample well; (g) sample is incubated at 40 °C for 1 h. Enzyme digests intact proteins into peptides; (h) and (i) as solvent begins to evaporate from sample well, additional solvent (7.5 μL) is aspirated from solvent well and added to sample well [ (h) and (i) are performed at 30 min and 1 h]. (j) Proteins are digested into peptides after 1 h. (k) Plate is transferred to HPLC autosampler and peptides are analyzed by LC MS/MS

    Techniques Used: Transferring, Incubation, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    27) Product Images from "Activation of diverse signaling pathways by oncogenic PIK3CA mutations"

    Article Title: Activation of diverse signaling pathways by oncogenic PIK3CA mutations

    Journal: Nature communications

    doi: 10.1038/ncomms5961

    Phosphorylation regulation patterns in MCF10A, Ex9-KI and J124-treated Ex9-KI cells. (a–d) Representative MS spectra of modulated phosphopeptides corresponding to each regulation pattern type are shown along with the phosphopeptide sequences. (e) Distribution of log 2 transformed intensity ratios of phosphorylation increased peptides (Ex9-KI vs. MCF10A, fold change > 1.5). The x-axis shows log 2 transformed phosphopeptide intensity ratios, and the y-axis shows the density. Blue represents the ratios of Ex9-KI to MCF10A cells while red represents the ratio of J124-treated Ex9-KI to MCF10A cells. The p-value calculated using a paired Student’s t-test comparing the two distributions was 2.2E-16. (f) Western blots to confirm the phosphorylation status of a subset of phosphoproteins using phospho-specific antibodies, along with Western blots using antibodies against total proteins. β-actin served as loading control. (g) The number of regulated proteins found in enriched signaling pathways (Modified Fisher Exact P-Value
    Figure Legend Snippet: Phosphorylation regulation patterns in MCF10A, Ex9-KI and J124-treated Ex9-KI cells. (a–d) Representative MS spectra of modulated phosphopeptides corresponding to each regulation pattern type are shown along with the phosphopeptide sequences. (e) Distribution of log 2 transformed intensity ratios of phosphorylation increased peptides (Ex9-KI vs. MCF10A, fold change > 1.5). The x-axis shows log 2 transformed phosphopeptide intensity ratios, and the y-axis shows the density. Blue represents the ratios of Ex9-KI to MCF10A cells while red represents the ratio of J124-treated Ex9-KI to MCF10A cells. The p-value calculated using a paired Student’s t-test comparing the two distributions was 2.2E-16. (f) Western blots to confirm the phosphorylation status of a subset of phosphoproteins using phospho-specific antibodies, along with Western blots using antibodies against total proteins. β-actin served as loading control. (g) The number of regulated proteins found in enriched signaling pathways (Modified Fisher Exact P-Value

    Techniques Used: Mass Spectrometry, Transformation Assay, Western Blot, Modification

    Widespread modulation of the kinome by PIK3CA mutants. (a) A phylogenetic tree (modified from Human Kinome Tree 58 ) of protein kinases identified in Ex9-KI cells. Phosphorylation increased kinases are in orange and kinases identified but did not changed in phosphorylation levels are in light green. A color-coded site regulation pattern is shown in the form of a circle divided into two parts. The top half represents the fold change of phosphorylation sites identified in Ex9-KI cells compared to MCF10A, whereas the bottom half represents the fold change ratio between J124-treated Ex9-KI cells compared to untreated cells. Regulated kinases that are known to be AKT substrates are underlined. (b) Significantly overrepresented linear phosphorylation motifs identified using Motif X program were indicated on the left of the panel. Phosphopeptides matching the motifs were used for prediction of their upstream kinases by NetworKIN program. Based on the total number of phospho-modulated peptides, percentage of the number of phosphopeptides as substrates of predicted kinases were calculated and demonstrated in the heatmap (right panel). (c) Sequence logos of overrepresented phosphoserine linear motifs enriched among the peptides whose phosphorylation levels were increased in Ex9-KI and/or Ex20-KI cells as compared to MCF10A cells.
    Figure Legend Snippet: Widespread modulation of the kinome by PIK3CA mutants. (a) A phylogenetic tree (modified from Human Kinome Tree 58 ) of protein kinases identified in Ex9-KI cells. Phosphorylation increased kinases are in orange and kinases identified but did not changed in phosphorylation levels are in light green. A color-coded site regulation pattern is shown in the form of a circle divided into two parts. The top half represents the fold change of phosphorylation sites identified in Ex9-KI cells compared to MCF10A, whereas the bottom half represents the fold change ratio between J124-treated Ex9-KI cells compared to untreated cells. Regulated kinases that are known to be AKT substrates are underlined. (b) Significantly overrepresented linear phosphorylation motifs identified using Motif X program were indicated on the left of the panel. Phosphopeptides matching the motifs were used for prediction of their upstream kinases by NetworKIN program. Based on the total number of phospho-modulated peptides, percentage of the number of phosphopeptides as substrates of predicted kinases were calculated and demonstrated in the heatmap (right panel). (c) Sequence logos of overrepresented phosphoserine linear motifs enriched among the peptides whose phosphorylation levels were increased in Ex9-KI and/or Ex20-KI cells as compared to MCF10A cells.

    Techniques Used: Modification, Sequencing

    28) Product Images from "Bpur, the Lyme Disease Spirochete's PUR Domain Protein"

    Article Title: Bpur, the Lyme Disease Spirochete's PUR Domain Protein

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.491357

    Illustrative examples of quantitative MS/MS data mined from limited proteolysis assay. All tryptic polypeptides, identified by a MASCOT search via Proteome Discoverer 1.3, were mapped against the linear amino acid sequence of Bpur for each time point and substrate group. Ion scores pertaining to individual polypeptide concentrations and the confidence of polypeptide identification through LTQ Velos Orbitrap mass coupled with a nano-LC Ultra/cHiPLC-nanoflex HPLC system are shown. Extracted ion chromatograms for polypeptides surrounding a given cleavage site were evaluated for signal intensity and peak area. Relative peak intensity (arbitrary units) is measured on the y axis, and peptide retention time (minutes) is measured on the x axis. Four representative data sets are presented. Black lines, no substrate added. Red lines, Bpur bound to dsDNA. Green lines, Bpur bound to ssDNA. Purple lines, Bpur bound to RNA. A, representative data set showing results when binding of a ligand did not change a rate of digestion, relative to the Bpur alone control reactions: TYFFNVK polypeptide presence after 20 min of incubation with trypsin. B, levels of the GDYFLNIVESK polypeptide after 10 min of incubation with trypsin. Binding of RNA or dsDNA inhibited cleavage adjacent to residue Lys-40. C, levels of the QKVSTGSVGSSAR polypeptide after 20 min of incubation with trypsin. To different extents, binding of each ligand inhibited cleavage at residue Lys-74. D, levels of the RSPSGDFERAIAVIK polypeptide after 20 min of incubation with trypsin. To different extents, each nucleic acid ligand decreased cleavage adjacent to residue Lys-40, resulting in the increased production of a larger polypeptide fragment.
    Figure Legend Snippet: Illustrative examples of quantitative MS/MS data mined from limited proteolysis assay. All tryptic polypeptides, identified by a MASCOT search via Proteome Discoverer 1.3, were mapped against the linear amino acid sequence of Bpur for each time point and substrate group. Ion scores pertaining to individual polypeptide concentrations and the confidence of polypeptide identification through LTQ Velos Orbitrap mass coupled with a nano-LC Ultra/cHiPLC-nanoflex HPLC system are shown. Extracted ion chromatograms for polypeptides surrounding a given cleavage site were evaluated for signal intensity and peak area. Relative peak intensity (arbitrary units) is measured on the y axis, and peptide retention time (minutes) is measured on the x axis. Four representative data sets are presented. Black lines, no substrate added. Red lines, Bpur bound to dsDNA. Green lines, Bpur bound to ssDNA. Purple lines, Bpur bound to RNA. A, representative data set showing results when binding of a ligand did not change a rate of digestion, relative to the Bpur alone control reactions: TYFFNVK polypeptide presence after 20 min of incubation with trypsin. B, levels of the GDYFLNIVESK polypeptide after 10 min of incubation with trypsin. Binding of RNA or dsDNA inhibited cleavage adjacent to residue Lys-40. C, levels of the QKVSTGSVGSSAR polypeptide after 20 min of incubation with trypsin. To different extents, binding of each ligand inhibited cleavage at residue Lys-74. D, levels of the RSPSGDFERAIAVIK polypeptide after 20 min of incubation with trypsin. To different extents, each nucleic acid ligand decreased cleavage adjacent to residue Lys-40, resulting in the increased production of a larger polypeptide fragment.

    Techniques Used: Mass Spectrometry, Proteolysis Assay, Sequencing, High Performance Liquid Chromatography, Binding Assay, Incubation

    29) Product Images from "Quantitative Proteomic Analysis of Gene Regulation by miR-34a and miR-34c"

    Article Title: Quantitative Proteomic Analysis of Gene Regulation by miR-34a and miR-34c

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092166

    Hypothetical model of the differential effect of miR-34a and miR-34c on the p53 feedback network integrating both known protein interactions with miR-34a and miR-34c and our observations. Grey color indicates proteins not quantified in our data but with known miR-34 interactions. Color indicates down-regulation (red) and up-regulation (green) of proteins quantified in our data. miR-34a has a positive feedback loop with p53 by blocking its inhibitor Sirt1. The effect of miR-34c on Sirt1 is not known. While miR-34a induction is heavily dependent on p53 levels, miR-34c expression can also be induced via alternative pathways (of which Mapk14 is depicted here). c-Myc is no target of miR-34a under normal expression conditions but is strongly repressed by miR-34c. This leads to inhibition of cell proliferation, DNA replication and induction of S-phase arrest. c-Myc also hinders apoptosis induction under p53 activation settings.
    Figure Legend Snippet: Hypothetical model of the differential effect of miR-34a and miR-34c on the p53 feedback network integrating both known protein interactions with miR-34a and miR-34c and our observations. Grey color indicates proteins not quantified in our data but with known miR-34 interactions. Color indicates down-regulation (red) and up-regulation (green) of proteins quantified in our data. miR-34a has a positive feedback loop with p53 by blocking its inhibitor Sirt1. The effect of miR-34c on Sirt1 is not known. While miR-34a induction is heavily dependent on p53 levels, miR-34c expression can also be induced via alternative pathways (of which Mapk14 is depicted here). c-Myc is no target of miR-34a under normal expression conditions but is strongly repressed by miR-34c. This leads to inhibition of cell proliferation, DNA replication and induction of S-phase arrest. c-Myc also hinders apoptosis induction under p53 activation settings.

    Techniques Used: Blocking Assay, Expressing, Inhibition, Activation Assay

    Functional enrichment analysis of miR-34a and miR-34c. KEGG pathway enrichment for subsets of miR-34a and miR-34c targets (for all proteins down-regulated log2
    Figure Legend Snippet: Functional enrichment analysis of miR-34a and miR-34c. KEGG pathway enrichment for subsets of miR-34a and miR-34c targets (for all proteins down-regulated log2

    Techniques Used: Functional Assay

    Distribution of chimera regulation within exclusive targets for either miR-34a or miR-34c. Shown are proteins exclusively regulated by miR-34a or miR-34c with a log2 fold change cutoff of ≤ –0.3. Colors indicate whether these exclusives targets are also regulated by any or both miR-34 chimeras mir-34ac and miR-34ca. The significance of the overlap between exclusive targets of the miRNAs and their chimeras was calculated using a hypergeometric test.
    Figure Legend Snippet: Distribution of chimera regulation within exclusive targets for either miR-34a or miR-34c. Shown are proteins exclusively regulated by miR-34a or miR-34c with a log2 fold change cutoff of ≤ –0.3. Colors indicate whether these exclusives targets are also regulated by any or both miR-34 chimeras mir-34ac and miR-34ca. The significance of the overlap between exclusive targets of the miRNAs and their chimeras was calculated using a hypergeometric test.

    Techniques Used:

    Proteomic comparison of miR-34a and miR-34c targets. ( A ) The correlation of log2 fold changes between miR-34a and miR-34c in the same transfection experiment (n = 2419) show a lower Spearman correlation than the two replicates of miR-34a (n = 1404) ( B ). This holds also true when comparing miR-34a experiments from different days (n = 1777) ( C ). Spearman coefficients for all proteins are marked in black, while seed containing proteins are indicated in red. ( D ) The overlap of common targets between miR-34a and miR-34c is rather low. ( E ) The overlap of miR-34a targets (–0.3 log2 FC) from SW480 cells [29] is bigger with miR-34a than with miR-34c targets in our HeLa dataset. Venn diagrams show the overlap of the 81 down-regulated proteins quantified in both the Sw480 and our HeLa dataset. Numbers in Venn diagrams depict total number of proteins down-regulated by log2
    Figure Legend Snippet: Proteomic comparison of miR-34a and miR-34c targets. ( A ) The correlation of log2 fold changes between miR-34a and miR-34c in the same transfection experiment (n = 2419) show a lower Spearman correlation than the two replicates of miR-34a (n = 1404) ( B ). This holds also true when comparing miR-34a experiments from different days (n = 1777) ( C ). Spearman coefficients for all proteins are marked in black, while seed containing proteins are indicated in red. ( D ) The overlap of common targets between miR-34a and miR-34c is rather low. ( E ) The overlap of miR-34a targets (–0.3 log2 FC) from SW480 cells [29] is bigger with miR-34a than with miR-34c targets in our HeLa dataset. Venn diagrams show the overlap of the 81 down-regulated proteins quantified in both the Sw480 and our HeLa dataset. Numbers in Venn diagrams depict total number of proteins down-regulated by log2

    Techniques Used: Transfection

    Experimental setup. A, Each member of the miR-34 family is transfected individually into HeLa cells in light SILAC medium. In parallel, a mock transfected control sample is prepared for each member. After 8h of transfection the samples are transferred to different SILAC medium, heavy (“H”) for the control and medium-heavy (“M”) for the miRNA transfected cells. After 24h of pulse labeling corresponding sample are combined and processed for mass spectrometry. The resulting peaks for one peptide are shown as an example. Peptides produced before pulse labeling appear as light peaks and can be disregarded. Differences in protein synthesis between control and miRNA-transfected samples can be read from the H/M ratio of the respective peptides. B , Nucleotide sequences of the miR-34 family members miR-34a and miR-34c. To investigate the importance of 5′ versus 3′ends two miRNA chimeras were constructed swapping head (nt 1-9) and tail of miR-34a and miR-34c respectively. Differences in the nucleotide sequence are marked in blue. The seed is labeled red.
    Figure Legend Snippet: Experimental setup. A, Each member of the miR-34 family is transfected individually into HeLa cells in light SILAC medium. In parallel, a mock transfected control sample is prepared for each member. After 8h of transfection the samples are transferred to different SILAC medium, heavy (“H”) for the control and medium-heavy (“M”) for the miRNA transfected cells. After 24h of pulse labeling corresponding sample are combined and processed for mass spectrometry. The resulting peaks for one peptide are shown as an example. Peptides produced before pulse labeling appear as light peaks and can be disregarded. Differences in protein synthesis between control and miRNA-transfected samples can be read from the H/M ratio of the respective peptides. B , Nucleotide sequences of the miR-34 family members miR-34a and miR-34c. To investigate the importance of 5′ versus 3′ends two miRNA chimeras were constructed swapping head (nt 1-9) and tail of miR-34a and miR-34c respectively. Differences in the nucleotide sequence are marked in blue. The seed is labeled red.

    Techniques Used: Transfection, Labeling, Mass Spectrometry, Produced, Construct, Sequencing

    Luciferase assays of specific miR-34a and miR-34c targets. Displayed is the relative protein production after transfection of miR-34a and miR-34c together with vector constructs containing the 3′ UTR of the two seed-containing miR-34a specific targets Fkbp8 ( A ) and Vcl ( B ) or the seedless miR-34c specific target Prkara2a ( C ). The SILAC change displays the difference of log2 fold changes as observed in the proteomic data. The known miR-34 target c-Met is used as control vector and miR-16 as control siRNA that does not significantly influence the levels of either target. Relative protein production for Prkar2a transfected with miR-34c were higher than depicted, reaching a 191%, which is indicated by dashed lines. P-values were calculated by one-tailed one-sample t-test from n = 3 biological replicates.
    Figure Legend Snippet: Luciferase assays of specific miR-34a and miR-34c targets. Displayed is the relative protein production after transfection of miR-34a and miR-34c together with vector constructs containing the 3′ UTR of the two seed-containing miR-34a specific targets Fkbp8 ( A ) and Vcl ( B ) or the seedless miR-34c specific target Prkara2a ( C ). The SILAC change displays the difference of log2 fold changes as observed in the proteomic data. The known miR-34 target c-Met is used as control vector and miR-16 as control siRNA that does not significantly influence the levels of either target. Relative protein production for Prkar2a transfected with miR-34c were higher than depicted, reaching a 191%, which is indicated by dashed lines. P-values were calculated by one-tailed one-sample t-test from n = 3 biological replicates.

    Techniques Used: Luciferase, Transfection, Plasmid Preparation, Construct, One-tailed Test

    MiR-34a and miR-34c repress synthesis of many proteins. ( A ) Known targets of the miR-34 family are down-regulated in our dataset (error bars indicate standard deviations from two or three experiments). ( B ) Cumulative distribution plots show that synthesis of proteins with miR-34 seed matches in their mRNA 3′UTRs is repressed by transfecting miR-34a (n = 4612). ( C ) The same holds true for the miR-34c transfection (n = 4094). ( D ) When selecting for the seed of miR-1 this correlation between seed and down-regulation is not visible (n = 4612). ( E ) Enrichment of seed matches in down-regulated proteins is significant even at mild log2FC cut-offs (hypergeometric test, dashed line: log2FC cut-off -0.3, dotted line: significance threshold p = 0.05, n = 4612). ( F ) Sylarray analysis [42] of miR-34a proteins sorted from down- (left) to up-regulated (right) renders the mature miR-34a seed (ACTGCC) as enriched nucleotide motif; however, also the *seed of our siRNA duplex (GCTGGT) is enriched in down-regulated proteins . ( G ) Similar observations are made for miR-34c. ( H ) Overview of the numbers of quantified as well as regulated proteins in miR-34a and miR-34c.
    Figure Legend Snippet: MiR-34a and miR-34c repress synthesis of many proteins. ( A ) Known targets of the miR-34 family are down-regulated in our dataset (error bars indicate standard deviations from two or three experiments). ( B ) Cumulative distribution plots show that synthesis of proteins with miR-34 seed matches in their mRNA 3′UTRs is repressed by transfecting miR-34a (n = 4612). ( C ) The same holds true for the miR-34c transfection (n = 4094). ( D ) When selecting for the seed of miR-1 this correlation between seed and down-regulation is not visible (n = 4612). ( E ) Enrichment of seed matches in down-regulated proteins is significant even at mild log2FC cut-offs (hypergeometric test, dashed line: log2FC cut-off -0.3, dotted line: significance threshold p = 0.05, n = 4612). ( F ) Sylarray analysis [42] of miR-34a proteins sorted from down- (left) to up-regulated (right) renders the mature miR-34a seed (ACTGCC) as enriched nucleotide motif; however, also the *seed of our siRNA duplex (GCTGGT) is enriched in down-regulated proteins . ( G ) Similar observations are made for miR-34c. ( H ) Overview of the numbers of quantified as well as regulated proteins in miR-34a and miR-34c.

    Techniques Used: Transfection

    30) Product Images from "Phosphoproteomic analysis of the antitumor effects of ginsenoside Rg3 in human breast cancer cells"

    Article Title: Phosphoproteomic analysis of the antitumor effects of ginsenoside Rg3 in human breast cancer cells

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.7654

    Schematic illustration of the TMT-based quantitative whole proteomic/phosphoproteomic pipeline. Cells treated with ginsenoside Rg3 for different time were subjected to filter-assisted sample preparation-based sample preparation. 6-plex TMT labeling and bRP-HPLC were performed and collected fractions were concatenated. A proportion of these fractions were subjected to TiO 2 enrichment. Enriched phosphopeptides, along with unprocessed peptides were analyzed using an Orbitrap-equipped mass spectrometer. TMT, tandem mass tag; LC-MS/MS, liquid chromatography tandem mass spectrometry; bRP-HPLC, basic reversed phase high-performance liquid chromatography; TiO 2 , titanium dioxide.
    Figure Legend Snippet: Schematic illustration of the TMT-based quantitative whole proteomic/phosphoproteomic pipeline. Cells treated with ginsenoside Rg3 for different time were subjected to filter-assisted sample preparation-based sample preparation. 6-plex TMT labeling and bRP-HPLC were performed and collected fractions were concatenated. A proportion of these fractions were subjected to TiO 2 enrichment. Enriched phosphopeptides, along with unprocessed peptides were analyzed using an Orbitrap-equipped mass spectrometer. TMT, tandem mass tag; LC-MS/MS, liquid chromatography tandem mass spectrometry; bRP-HPLC, basic reversed phase high-performance liquid chromatography; TiO 2 , titanium dioxide.

    Techniques Used: Sample Prep, Labeling, High Performance Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography

    31) Product Images from "Development of a sequential workflow based on LC-PRM for the verification of endometrial cancer protein biomarkers in uterine aspirate samples"

    Article Title: Development of a sequential workflow based on LC-PRM for the verification of endometrial cancer protein biomarkers in uterine aspirate samples

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10632

    Effect of blood content on biomarker candidate detection Experimental design and examples of concentration profiles of 3 potential biomarkers showing increasing and 3 decreasing profiles when uterine aspirate is diluted by increasing amount of full blood. The 32 candidates showing an increasing profile were rejected for further steps in the study.
    Figure Legend Snippet: Effect of blood content on biomarker candidate detection Experimental design and examples of concentration profiles of 3 potential biomarkers showing increasing and 3 decreasing profiles when uterine aspirate is diluted by increasing amount of full blood. The 32 candidates showing an increasing profile were rejected for further steps in the study.

    Techniques Used: Biomarker Assay, Concentration Assay

    Scattering plots of the abundance of 17 peptides coming from 10 biomarkers in the verification study Scattering plots depicting the distribution of the light/heavy (L/H) ratios across the 20 EC patients and 18 controls of the best individual performing peptides (AUC > 0.9) belonging to 10 biomarkers.
    Figure Legend Snippet: Scattering plots of the abundance of 17 peptides coming from 10 biomarkers in the verification study Scattering plots depicting the distribution of the light/heavy (L/H) ratios across the 20 EC patients and 18 controls of the best individual performing peptides (AUC > 0.9) belonging to 10 biomarkers.

    Techniques Used:

    32) Product Images from "Exosomes Derived from Human Primed Mesenchymal Stem Cells Induce Mitosis and Potentiate Growth Factor Secretion"

    Article Title: Exosomes Derived from Human Primed Mesenchymal Stem Cells Induce Mitosis and Potentiate Growth Factor Secretion

    Journal: Stem Cells and Development

    doi: 10.1089/scd.2018.0200

    Flow cytometry, HiRIEF LC-MS/MS proteomics, nanoparticle tracking analysis and electron microscopy analysis of pMSCs and pMEXs. (A–C) Flow cytometry analysis of MSC surface marker expression using monoclonal primary conjugated antibodies against the canonical MSC markers CD73, CD90, and CD105. (D) Nanoparticle tracking analysis determined the size distribution of pMEX, with a mean diameter of 163 nm, red highlight = distribution of events, black line = median. (E) Transmission electron microscopy of pMEX with uranyl acetate negative staining (scale bar 200 nm). (F) HiRIEF LC-MS/MS proteomic analysis identified 93 and 94 exosomal markers out of the top 100 most cited in the ExoCarta database in pMSCs and pMEXs, respectively. (G) Of all, 6.7% proteins observed in pMEX were exclusively detected within pMEX, whereas 71.8% of all pMSCs proteins were exclusively detected in pMSCs. (H) FBS-derived proteins were detected in both pMSC and pMEX ( n = 3/group, FDR 1%). FBS, fetal bovine serum; pMSCs, primed mesenchymal stem cells.
    Figure Legend Snippet: Flow cytometry, HiRIEF LC-MS/MS proteomics, nanoparticle tracking analysis and electron microscopy analysis of pMSCs and pMEXs. (A–C) Flow cytometry analysis of MSC surface marker expression using monoclonal primary conjugated antibodies against the canonical MSC markers CD73, CD90, and CD105. (D) Nanoparticle tracking analysis determined the size distribution of pMEX, with a mean diameter of 163 nm, red highlight = distribution of events, black line = median. (E) Transmission electron microscopy of pMEX with uranyl acetate negative staining (scale bar 200 nm). (F) HiRIEF LC-MS/MS proteomic analysis identified 93 and 94 exosomal markers out of the top 100 most cited in the ExoCarta database in pMSCs and pMEXs, respectively. (G) Of all, 6.7% proteins observed in pMEX were exclusively detected within pMEX, whereas 71.8% of all pMSCs proteins were exclusively detected in pMSCs. (H) FBS-derived proteins were detected in both pMSC and pMEX ( n = 3/group, FDR 1%). FBS, fetal bovine serum; pMSCs, primed mesenchymal stem cells.

    Techniques Used: Flow Cytometry, Cytometry, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Electron Microscopy, Marker, Expressing, Transmission Assay, Negative Staining, Derivative Assay

    33) Product Images from "Protein O-mannosylation deficiency increases LprG-associated lipoarabinomannan release by Mycobacterium tuberculosis and enhances the TLR2-associated inflammatory response"

    Article Title: Protein O-mannosylation deficiency increases LprG-associated lipoarabinomannan release by Mycobacterium tuberculosis and enhances the TLR2-associated inflammatory response

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08489-7

    Impact of LprG O -mannosylation on the release of the LprG/LAM complex in M . smegmatis . Similar amounts of LprG-His or LprG-His T231A purified from either culture medium or bacterial cells were separated by SDS-PAGE, transferred onto membranes and revealed using anti-LAM or anti-His antibodies. The genetic backgrounds in which the protein was produced are indicated. The upper band revealed by the anti-His antibody is a non-specific cross-reacting protein. The signal intensity for LAM was quantified and plotted in the graph below the Western blot image. This blot is representative of three independent experiments.
    Figure Legend Snippet: Impact of LprG O -mannosylation on the release of the LprG/LAM complex in M . smegmatis . Similar amounts of LprG-His or LprG-His T231A purified from either culture medium or bacterial cells were separated by SDS-PAGE, transferred onto membranes and revealed using anti-LAM or anti-His antibodies. The genetic backgrounds in which the protein was produced are indicated. The upper band revealed by the anti-His antibody is a non-specific cross-reacting protein. The signal intensity for LAM was quantified and plotted in the graph below the Western blot image. This blot is representative of three independent experiments.

    Techniques Used: Laser Capture Microdissection, Purification, SDS Page, Produced, Western Blot

    MS analyses of LprG and LprG T231A glycosylation. ( A ) Deconvoluted ESI-HRMS spectrum of the purified recombinant LprG-His expressed in M . tuberculosis . ( B ) Peptide sequence of the molecular species observed in A reporting the fragment ions detected in the top-down electron transfer dissociation spectrum of the fragmentation of the 22,541 Da molecular mass ion precursor allowing the localization of the unique hexose on the T231. ( C ) Deconvoluted ESI-HRMS spectrum of purified recombinant LprG-His T231A expressed in M . smegmatis demonstrating the absence of glycosylation of the mutated protein. ( D ) Peptide sequence of the molecular species observed in C.
    Figure Legend Snippet: MS analyses of LprG and LprG T231A glycosylation. ( A ) Deconvoluted ESI-HRMS spectrum of the purified recombinant LprG-His expressed in M . tuberculosis . ( B ) Peptide sequence of the molecular species observed in A reporting the fragment ions detected in the top-down electron transfer dissociation spectrum of the fragmentation of the 22,541 Da molecular mass ion precursor allowing the localization of the unique hexose on the T231. ( C ) Deconvoluted ESI-HRMS spectrum of purified recombinant LprG-His T231A expressed in M . smegmatis demonstrating the absence of glycosylation of the mutated protein. ( D ) Peptide sequence of the molecular species observed in C.

    Techniques Used: Mass Spectrometry, Purification, Recombinant, Sequencing

    34) Product Images from "Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes reveal genes and pathways involved in osteoarthritis"

    Article Title: Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes reveal genes and pathways involved in osteoarthritis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09335-6

    Comparison of changes identified in the –omics experiments. ( a ) Comparison of the log-fold-changes between all genes identified in both the proteomics and RNA-seq experiments. Each gene is represented as a single point, and the colour corresponds to whether the gene is identified as differentially expressed using edgeR in the RNA-seq or proteomics experiments, or both. The trend lines are derived from a linear regression in each subset. Positive fold changes indicate increased expression in degraded samples. ( b ) Comparison of RNA-seq log-fold-change and mean promoter region methylation change. The trend lines are derived from a linear regression in each subset. Genes are coloured according to the results of the RNA-seq and the promoter-region analyses analogously to Fig. 2a.
    Figure Legend Snippet: Comparison of changes identified in the –omics experiments. ( a ) Comparison of the log-fold-changes between all genes identified in both the proteomics and RNA-seq experiments. Each gene is represented as a single point, and the colour corresponds to whether the gene is identified as differentially expressed using edgeR in the RNA-seq or proteomics experiments, or both. The trend lines are derived from a linear regression in each subset. Positive fold changes indicate increased expression in degraded samples. ( b ) Comparison of RNA-seq log-fold-change and mean promoter region methylation change. The trend lines are derived from a linear regression in each subset. Genes are coloured according to the results of the RNA-seq and the promoter-region analyses analogously to Fig. 2a.

    Techniques Used: RNA Sequencing Assay, Derivative Assay, Expressing, Methylation

    35) Product Images from "Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis"

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007117

    GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.
    Figure Legend Snippet: GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Techniques Used: Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Blocking Assay, Silver Staining, Quantitation Assay, Western Blot, Imaging, Incubation

    Models for p12-chromatin binding. (A) Proposed model for the different functions of p12. The p12 region of Gag and p12 protein in the viral PIC differ in their affinity for cellular proteins and chromatin. We propose that as part of Gag, or when expressed as a recombinant GST-fusion protein, p12 exists in an unstructured conformation with low affinity for nucleosomes but relatively high affinity for host proteins such as clathrin and NEDD4-like E3 ligases which facilitate late replication events. Following Gag cleavage, the binding of the p12 NTD to the CA lattice promotes a change in the conformation of p12 which increases the affinity of the p12 CTD for nucleosomes. During mitosis, the breakdown of the nuclear envelope allows the p12/CA-containing PIC to access chromatin. The PIC is targeted to nucleosomes on mitotic chromatin by CA-bound, phosphorylated p12. Exit from mitosis promotes the de-phosphorylation of p12 and the dissociation of p12 and CA from chromatin. BET proteins can then bind IN and direct the viral cDNA to gene promoter regions where integration occurs. (B) Proposed relationship between virus infectivity and affinity of p12 for chromatin. We suggest that the affinity of p12 for chromatin is fine-tuned for optimal infectivity with deviations incurring a fitness cost. Mutations in p12 that increase or decrease chromatin binding (measured, in blue, or extrapolated, in red) alter viral infectivity as shown on the left. Only interactions above an arbitrary threshold can be detected by GST-pull down assays.
    Figure Legend Snippet: Models for p12-chromatin binding. (A) Proposed model for the different functions of p12. The p12 region of Gag and p12 protein in the viral PIC differ in their affinity for cellular proteins and chromatin. We propose that as part of Gag, or when expressed as a recombinant GST-fusion protein, p12 exists in an unstructured conformation with low affinity for nucleosomes but relatively high affinity for host proteins such as clathrin and NEDD4-like E3 ligases which facilitate late replication events. Following Gag cleavage, the binding of the p12 NTD to the CA lattice promotes a change in the conformation of p12 which increases the affinity of the p12 CTD for nucleosomes. During mitosis, the breakdown of the nuclear envelope allows the p12/CA-containing PIC to access chromatin. The PIC is targeted to nucleosomes on mitotic chromatin by CA-bound, phosphorylated p12. Exit from mitosis promotes the de-phosphorylation of p12 and the dissociation of p12 and CA from chromatin. BET proteins can then bind IN and direct the viral cDNA to gene promoter regions where integration occurs. (B) Proposed relationship between virus infectivity and affinity of p12 for chromatin. We suggest that the affinity of p12 for chromatin is fine-tuned for optimal infectivity with deviations incurring a fitness cost. Mutations in p12 that increase or decrease chromatin binding (measured, in blue, or extrapolated, in red) alter viral infectivity as shown on the left. Only interactions above an arbitrary threshold can be detected by GST-pull down assays.

    Techniques Used: Binding Assay, Recombinant, De-Phosphorylation Assay, Infection

    GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).
    Figure Legend Snippet: GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Techniques Used: Expressing, Lysis, Incubation, SDS Page, Staining, Silver Staining, Mutagenesis, Infection, Activity Assay, Reporter Assay

    36) Product Images from "Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates *"

    Article Title: Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M115.055384

    The majority of uncharacterized false positive methyl-PSMs in S. cerevisiae cell lysate samples are predicted by decoy database searches in ETD- and CID-derived datasets but not in HCD-derived datasets. A , proportions of total non-redundant false positive methyl-PSMs that can be explained by equal or higher scoring PSMs with alternative sites of arginine or lysine methylation, cysteinyl- S -β-propionamide, or methylated glutamic or aspartic acid residues, as shown for combined SDS-PAGE (Coomassie stained and unstained)- and HILIC-derived datasets. Remaining false positive methyl-PSMs are considered uncharacterized. Proportions of uncharacterized false positive methyl-PSMs predicted by the target-decoy approach (using separate methyl-PSM FDR estimates) are shown. B , average amino acid compositions of decoy lysine and arginine methyl-PSMs relative to respective high confidence unmethylated lysine- and arginine-containing PSMs (above), and the difference between the average amino acid compositions of decoy methyl-PSMs and uncharacterized false positive PSMs from target database searches (below). For each amino acid, numbers of mass differentials between single amino acids that match to mass differentials associated with mono-, di-, or tri-methylation (14.0157, 28.0314, and 42.0470 Da, respectively) are listed. Amino acids with no such mass differentials are under-represented in decoy methyl-PSMs ( light gray boxes ). ETD data were from an LTQ Orbitrap Velos Pro ETD instrument; CID data were from an LTQ Orbitrap Velos Pro instrument; HCD data were from a Q Exactive Plus instrument. All data are from PSMs of Mascot Except value of
    Figure Legend Snippet: The majority of uncharacterized false positive methyl-PSMs in S. cerevisiae cell lysate samples are predicted by decoy database searches in ETD- and CID-derived datasets but not in HCD-derived datasets. A , proportions of total non-redundant false positive methyl-PSMs that can be explained by equal or higher scoring PSMs with alternative sites of arginine or lysine methylation, cysteinyl- S -β-propionamide, or methylated glutamic or aspartic acid residues, as shown for combined SDS-PAGE (Coomassie stained and unstained)- and HILIC-derived datasets. Remaining false positive methyl-PSMs are considered uncharacterized. Proportions of uncharacterized false positive methyl-PSMs predicted by the target-decoy approach (using separate methyl-PSM FDR estimates) are shown. B , average amino acid compositions of decoy lysine and arginine methyl-PSMs relative to respective high confidence unmethylated lysine- and arginine-containing PSMs (above), and the difference between the average amino acid compositions of decoy methyl-PSMs and uncharacterized false positive PSMs from target database searches (below). For each amino acid, numbers of mass differentials between single amino acids that match to mass differentials associated with mono-, di-, or tri-methylation (14.0157, 28.0314, and 42.0470 Da, respectively) are listed. Amino acids with no such mass differentials are under-represented in decoy methyl-PSMs ( light gray boxes ). ETD data were from an LTQ Orbitrap Velos Pro ETD instrument; CID data were from an LTQ Orbitrap Velos Pro instrument; HCD data were from a Q Exactive Plus instrument. All data are from PSMs of Mascot Except value of

    Techniques Used: Derivative Assay, Methylation, SDS Page, Staining, Hydrophilic Interaction Liquid Chromatography

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

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

    Journal: Chemical research in toxicology

    doi: 10.1021/acs.chemrestox.6b00416

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

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

    38) Product Images from "ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma"

    Article Title: ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20145450

    Proteomic and genomic workflows Cell lysates from control samples and samples derived from 1 and 3 days after PLX4720 treatment were digested with Lys-C/Trypsin, labeled by triplex dimethyl approach and mixed in 1:1:1 ratios. For protein expression analysis, 200 μg of digested lysate was fractionated by SCX and each fraction was analyzed by LC/MS/MS to determine the relative protein expression levels for every time point compared to the control. For the phosphoproteome, 3 mg of digested lysate was fractionated by SCX and each fraction was enriched for phosphopeptides by Ti 4+ -IMAC prior to LC/MS/MS analysis. Melanoma cells were transduced with a lentiviral kinome library, containing ˜4,000 shRNAs targeting ˜500 kinases. Cells were treated either with DMSO (control) or with BRAFi or ERKi. Genomic DNA was isolated, and hairpins were amplified by PCR. Using deep sequencing, the hairpins that specifically dropped out in the treated sample were identified. In this case, the absence of the blue bar in deep sequencing indicates schematically a synthetic lethal effect of the shRNA and BRAFi/ERKi.
    Figure Legend Snippet: Proteomic and genomic workflows Cell lysates from control samples and samples derived from 1 and 3 days after PLX4720 treatment were digested with Lys-C/Trypsin, labeled by triplex dimethyl approach and mixed in 1:1:1 ratios. For protein expression analysis, 200 μg of digested lysate was fractionated by SCX and each fraction was analyzed by LC/MS/MS to determine the relative protein expression levels for every time point compared to the control. For the phosphoproteome, 3 mg of digested lysate was fractionated by SCX and each fraction was enriched for phosphopeptides by Ti 4+ -IMAC prior to LC/MS/MS analysis. Melanoma cells were transduced with a lentiviral kinome library, containing ˜4,000 shRNAs targeting ˜500 kinases. Cells were treated either with DMSO (control) or with BRAFi or ERKi. Genomic DNA was isolated, and hairpins were amplified by PCR. Using deep sequencing, the hairpins that specifically dropped out in the treated sample were identified. In this case, the absence of the blue bar in deep sequencing indicates schematically a synthetic lethal effect of the shRNA and BRAFi/ERKi.

    Techniques Used: Derivative Assay, Labeling, Expressing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Transduction, Isolation, Amplification, Polymerase Chain Reaction, Sequencing, shRNA

    39) Product Images from "Extensive Post-translational Modification of Active and Inactivated Forms of Endogenous p53 *"

    Article Title: Extensive Post-translational Modification of Active and Inactivated Forms of Endogenous p53 *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M113.030254

    Representative MS/MS spectra of Ε p53. Peptides prepared from p53 isolated from HFFs treated with etoposide were subjected to reversed-phase nano-LC-MS and MS/MS on a UPLC-Orbitrap Velos platform as described under “Experimental Procedures.”
    Figure Legend Snippet: Representative MS/MS spectra of Ε p53. Peptides prepared from p53 isolated from HFFs treated with etoposide were subjected to reversed-phase nano-LC-MS and MS/MS on a UPLC-Orbitrap Velos platform as described under “Experimental Procedures.”

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

    Spectral count profiles of modified Ser, Thr, and Arg residues. Peptides from equivalent populations of ΔE1B p53 and E p53 isolated in parallel were subjected to nano-UPLC-MS and MS/MS analyses on the Orbitrap Velos platform. Modification profiles
    Figure Legend Snippet: Spectral count profiles of modified Ser, Thr, and Arg residues. Peptides from equivalent populations of ΔE1B p53 and E p53 isolated in parallel were subjected to nano-UPLC-MS and MS/MS analyses on the Orbitrap Velos platform. Modification profiles

    Techniques Used: Modification, Isolation, Mass Spectrometry

    Representative MS/MS spectra of ΔE1B p53. Peptides prepared from p53 isolates were subjected to reversed-phase nano-LC-MS and MS/MS on a UPLC-Orbitrap Velos platform as described under “Experimental Procedures.” Shown are representative
    Figure Legend Snippet: Representative MS/MS spectra of ΔE1B p53. Peptides prepared from p53 isolates were subjected to reversed-phase nano-LC-MS and MS/MS on a UPLC-Orbitrap Velos platform as described under “Experimental Procedures.” Shown are representative

    Techniques Used: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    Representative MS/MS spectra of COS-1 p53. Peptides prepared from p53 isolated from COS-1 cells were subjected to reversed-phase nano-LC-MS and MS/MS on a UPLC-Orbitrap Velos platform as described under “Experimental Procedures.” Shown
    Figure Legend Snippet: Representative MS/MS spectra of COS-1 p53. Peptides prepared from p53 isolated from COS-1 cells were subjected to reversed-phase nano-LC-MS and MS/MS on a UPLC-Orbitrap Velos platform as described under “Experimental Procedures.” Shown

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

    40) Product Images from "Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates *"

    Article Title: Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M115.055384

    The majority of uncharacterized false positive methyl-PSMs in S. cerevisiae cell lysate samples are predicted by decoy database searches in ETD- and CID-derived datasets but not in HCD-derived datasets. A , proportions of total non-redundant false positive methyl-PSMs that can be explained by equal or higher scoring PSMs with alternative sites of arginine or lysine methylation, cysteinyl- S -β-propionamide, or methylated glutamic or aspartic acid residues, as shown for combined SDS-PAGE (Coomassie stained and unstained)- and HILIC-derived datasets. Remaining false positive methyl-PSMs are considered uncharacterized. Proportions of uncharacterized false positive methyl-PSMs predicted by the target-decoy approach (using separate methyl-PSM FDR estimates) are shown. B , average amino acid compositions of decoy lysine and arginine methyl-PSMs relative to respective high confidence unmethylated lysine- and arginine-containing PSMs (above), and the difference between the average amino acid compositions of decoy methyl-PSMs and uncharacterized false positive PSMs from target database searches (below). For each amino acid, numbers of mass differentials between single amino acids that match to mass differentials associated with mono-, di-, or tri-methylation (14.0157, 28.0314, and 42.0470 Da, respectively) are listed. Amino acids with no such mass differentials are under-represented in decoy methyl-PSMs ( light gray boxes ). ETD data were from an LTQ Orbitrap Velos Pro ETD instrument; CID data were from an LTQ Orbitrap Velos Pro instrument; HCD data were from a Q Exactive Plus instrument. All data are from PSMs of Mascot Except value of
    Figure Legend Snippet: The majority of uncharacterized false positive methyl-PSMs in S. cerevisiae cell lysate samples are predicted by decoy database searches in ETD- and CID-derived datasets but not in HCD-derived datasets. A , proportions of total non-redundant false positive methyl-PSMs that can be explained by equal or higher scoring PSMs with alternative sites of arginine or lysine methylation, cysteinyl- S -β-propionamide, or methylated glutamic or aspartic acid residues, as shown for combined SDS-PAGE (Coomassie stained and unstained)- and HILIC-derived datasets. Remaining false positive methyl-PSMs are considered uncharacterized. Proportions of uncharacterized false positive methyl-PSMs predicted by the target-decoy approach (using separate methyl-PSM FDR estimates) are shown. B , average amino acid compositions of decoy lysine and arginine methyl-PSMs relative to respective high confidence unmethylated lysine- and arginine-containing PSMs (above), and the difference between the average amino acid compositions of decoy methyl-PSMs and uncharacterized false positive PSMs from target database searches (below). For each amino acid, numbers of mass differentials between single amino acids that match to mass differentials associated with mono-, di-, or tri-methylation (14.0157, 28.0314, and 42.0470 Da, respectively) are listed. Amino acids with no such mass differentials are under-represented in decoy methyl-PSMs ( light gray boxes ). ETD data were from an LTQ Orbitrap Velos Pro ETD instrument; CID data were from an LTQ Orbitrap Velos Pro instrument; HCD data were from a Q Exactive Plus instrument. All data are from PSMs of Mascot Except value of

    Techniques Used: Derivative Assay, Methylation, SDS Page, Staining, Hydrophilic Interaction Liquid Chromatography

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    Concentration Assay:

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    Thermo Fisher thermo orbitrap velos
    Characterization of the modification to the monomeric species: a) Sequence coverage of AqpZ from the Lys-C, trypsin and chymotrypsin digestions. Positive sequence identification is underlined; b) Targeted CID spectra of both the unmodified N-terminal peptide from the tryptic digest and; (c) Targeted CID spectra of both the modified N-terminal peptide from the tryptic digest. Both spectra were obtained on the <t>OrbiTrap</t> <t>Velos.</t>
    Thermo Orbitrap Velos, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ltq orbitrap velos pro mass spectrometer
    Identification of linoleic acid in the free fatty acid fraction purified from canine stratum corneum by APCI LC-MS. RSLC Dionex-U3000 coupled to <t>LTQ-Orbitrap</t> <t>Velos</t> Pro, Thermofisher Scientific with an APCI source. a Free fatty acids spectra. b Linoleic acid spectra- standard use
    Ltq Orbitrap Velos Pro Mass Spectrometer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher ltq orbitrap velos
    Tc-STAMS2 was tested for its robustness towards technical and experimental variations. Initially, the Tc-STAMS2 approach was tested for inter-laboratory comparison. Two unknown T . cruzi strains (A and B) were processed as described in the step B of Fig 1 and acquired using the EasynLC coupled to <t>LTQ-Orbitrap</t> <t>Velos</t> mass spectrometer located in the CEFAP mass spectrometry facility at the University of Sao Pa ulo, Sao Paulo, Brazil. The MS/MS spectral library was built using Sylvio X10 cl1 (DTU-I), Y (DTU-II), M6241 cl6 (DTU-III), CanIII cl1 (DTU-IV), MN cl2 (DTU-V), CL Brener (DTU-VI) and acquired in the PR group, Odense, Denmark using a similar LC-MS/MS setup (EasynLC coupled to LTQ-Orbitrap Velos). A1 and A2 indicate a biological duplicate of T . cruzi M6241 cl6 (DTU-III). B is the T . cruzi Sylvio X10 cl1 (DTU-I). Different sample preparation strategies were used to test the robustness of the Tc-STAMS2 approach such as changing the pH for peptide desalting. B/acid refers to peptides derived from sample B were purified using acidic conditions (0.1% TFA). B/basic refers to peptides derived from sample B were purified using basic conditions (0.1% ammonia). Moreover, different analytical parameters were changed in order to test the robustness of the Tc-STAMS2 approach such as the MS/MS fragmentation type, CID—Collision-Induced Dissociation and HCD—Higher-energy collisional dissociation. Different sample amounts were loaded onto the nano LC column. High and Low indicate 1 and 0.5 ug, respectively. The Tc-STAMS2 approach was robust towards different analytical and experimental challenges.
    Ltq Orbitrap Velos, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 490 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ltq orbitrap velos mass analyzer
    Differential phosphoproteomics between parental (HuH-7) cells and cells with acquired resistance to sorafenib (HuH-7 R ). a Workflow for quantitative phosphoproteomic analyses between parental (HuH-7) and HCC cells with acquired sorafenib resistance (HuH-7 R ) via SILAC-based mass spectrometry. Heavy and light cell lysates were mixed and digested with trypsin and fractionated by high-pH reverse-phase chromatography. Phosphopeptides were then purified with TiO 2 column and analyzed in an <t>LTQ-Orbitrap</t> <t>Velos</t> hybrid mass spectrometer. b Clustered gene ontology functional enrichment was assessed with DAVID. Upregulated phosphoproteins in HuH-7 R cells showing a SILAC fold change H/L ≥ 1.5 were analyzed. The top three functional clusters are listed. −Log ( p values) and enrichment scores are presented. c Pathway enrichment analysis of the upregulated phosphoproteins in HuH-7 R cells based on the KEGG pathway database with DAVID. Pathways with p values
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    Characterization of the modification to the monomeric species: a) Sequence coverage of AqpZ from the Lys-C, trypsin and chymotrypsin digestions. Positive sequence identification is underlined; b) Targeted CID spectra of both the unmodified N-terminal peptide from the tryptic digest and; (c) Targeted CID spectra of both the modified N-terminal peptide from the tryptic digest. Both spectra were obtained on the OrbiTrap Velos.

    Journal: Journal of the American Society for Mass Spectrometry

    Article Title: Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry as a Platform for Characterizing Multimeric Membrane Protein Complexes

    doi: 10.1007/s13361-017-1799-4

    Figure Lengend Snippet: Characterization of the modification to the monomeric species: a) Sequence coverage of AqpZ from the Lys-C, trypsin and chymotrypsin digestions. Positive sequence identification is underlined; b) Targeted CID spectra of both the unmodified N-terminal peptide from the tryptic digest and; (c) Targeted CID spectra of both the modified N-terminal peptide from the tryptic digest. Both spectra were obtained on the OrbiTrap Velos.

    Article Snippet: The proteolytic digests were measured by LC-MS/MS using a Waters NanoAcquity LC and LC-MS/MS interfaced to a Thermo OrbiTrap Velos MS (Thermo Scientific; Waltham, MA).

    Techniques: Modification, Sequencing

    Identification of linoleic acid in the free fatty acid fraction purified from canine stratum corneum by APCI LC-MS. RSLC Dionex-U3000 coupled to LTQ-Orbitrap Velos Pro, Thermofisher Scientific with an APCI source. a Free fatty acids spectra. b Linoleic acid spectra- standard use

    Journal: Archives of Dermatological Research

    Article Title: Linoleate-enriched diet increases both linoleic acid esterified to omega hydroxy very long chain fatty acids and free ceramides of canine stratum corneum without effect on protein-bound ceramides and skin barrier function

    doi: 10.1007/s00403-018-1845-5

    Figure Lengend Snippet: Identification of linoleic acid in the free fatty acid fraction purified from canine stratum corneum by APCI LC-MS. RSLC Dionex-U3000 coupled to LTQ-Orbitrap Velos Pro, Thermofisher Scientific with an APCI source. a Free fatty acids spectra. b Linoleic acid spectra- standard use

    Article Snippet: A 5 µl sample was injected in a RSLC Dionex-U300 column coupled to a LTQ-Orbitrap Velos Pro mass spectrometer (Thermofisher Scientific, Villebon-sur-Yvette, France) equipped with an atmospheric-pressure chemical ionization (APCI) interface.

    Techniques: Purification, Liquid Chromatography with Mass Spectroscopy

    Tc-STAMS2 was tested for its robustness towards technical and experimental variations. Initially, the Tc-STAMS2 approach was tested for inter-laboratory comparison. Two unknown T . cruzi strains (A and B) were processed as described in the step B of Fig 1 and acquired using the EasynLC coupled to LTQ-Orbitrap Velos mass spectrometer located in the CEFAP mass spectrometry facility at the University of Sao Pa ulo, Sao Paulo, Brazil. The MS/MS spectral library was built using Sylvio X10 cl1 (DTU-I), Y (DTU-II), M6241 cl6 (DTU-III), CanIII cl1 (DTU-IV), MN cl2 (DTU-V), CL Brener (DTU-VI) and acquired in the PR group, Odense, Denmark using a similar LC-MS/MS setup (EasynLC coupled to LTQ-Orbitrap Velos). A1 and A2 indicate a biological duplicate of T . cruzi M6241 cl6 (DTU-III). B is the T . cruzi Sylvio X10 cl1 (DTU-I). Different sample preparation strategies were used to test the robustness of the Tc-STAMS2 approach such as changing the pH for peptide desalting. B/acid refers to peptides derived from sample B were purified using acidic conditions (0.1% TFA). B/basic refers to peptides derived from sample B were purified using basic conditions (0.1% ammonia). Moreover, different analytical parameters were changed in order to test the robustness of the Tc-STAMS2 approach such as the MS/MS fragmentation type, CID—Collision-Induced Dissociation and HCD—Higher-energy collisional dissociation. Different sample amounts were loaded onto the nano LC column. High and Low indicate 1 and 0.5 ug, respectively. The Tc-STAMS2 approach was robust towards different analytical and experimental challenges.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Development of a Trypanosoma cruzi strain typing assay using MS2 peptide spectral libraries (Tc-STAMS2)

    doi: 10.1371/journal.pntd.0006351

    Figure Lengend Snippet: Tc-STAMS2 was tested for its robustness towards technical and experimental variations. Initially, the Tc-STAMS2 approach was tested for inter-laboratory comparison. Two unknown T . cruzi strains (A and B) were processed as described in the step B of Fig 1 and acquired using the EasynLC coupled to LTQ-Orbitrap Velos mass spectrometer located in the CEFAP mass spectrometry facility at the University of Sao Pa ulo, Sao Paulo, Brazil. The MS/MS spectral library was built using Sylvio X10 cl1 (DTU-I), Y (DTU-II), M6241 cl6 (DTU-III), CanIII cl1 (DTU-IV), MN cl2 (DTU-V), CL Brener (DTU-VI) and acquired in the PR group, Odense, Denmark using a similar LC-MS/MS setup (EasynLC coupled to LTQ-Orbitrap Velos). A1 and A2 indicate a biological duplicate of T . cruzi M6241 cl6 (DTU-III). B is the T . cruzi Sylvio X10 cl1 (DTU-I). Different sample preparation strategies were used to test the robustness of the Tc-STAMS2 approach such as changing the pH for peptide desalting. B/acid refers to peptides derived from sample B were purified using acidic conditions (0.1% TFA). B/basic refers to peptides derived from sample B were purified using basic conditions (0.1% ammonia). Moreover, different analytical parameters were changed in order to test the robustness of the Tc-STAMS2 approach such as the MS/MS fragmentation type, CID—Collision-Induced Dissociation and HCD—Higher-energy collisional dissociation. Different sample amounts were loaded onto the nano LC column. High and Low indicate 1 and 0.5 ug, respectively. The Tc-STAMS2 approach was robust towards different analytical and experimental challenges.

    Article Snippet: The MS analysis was performed using the LTQ-Orbitrap Velos (Thermo Scientific, Bremen, Germany).

    Techniques: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Sample Prep, Derivative Assay, Purification

    Differential phosphoproteomics between parental (HuH-7) cells and cells with acquired resistance to sorafenib (HuH-7 R ). a Workflow for quantitative phosphoproteomic analyses between parental (HuH-7) and HCC cells with acquired sorafenib resistance (HuH-7 R ) via SILAC-based mass spectrometry. Heavy and light cell lysates were mixed and digested with trypsin and fractionated by high-pH reverse-phase chromatography. Phosphopeptides were then purified with TiO 2 column and analyzed in an LTQ-Orbitrap Velos hybrid mass spectrometer. b Clustered gene ontology functional enrichment was assessed with DAVID. Upregulated phosphoproteins in HuH-7 R cells showing a SILAC fold change H/L ≥ 1.5 were analyzed. The top three functional clusters are listed. −Log ( p values) and enrichment scores are presented. c Pathway enrichment analysis of the upregulated phosphoproteins in HuH-7 R cells based on the KEGG pathway database with DAVID. Pathways with p values

    Journal: Experimental & Molecular Medicine

    Article Title: Quantitative phosphoproteomic analysis identifies the potential therapeutic target EphA2 for overcoming sorafenib resistance in hepatocellular carcinoma cells

    doi: 10.1038/s12276-020-0404-2

    Figure Lengend Snippet: Differential phosphoproteomics between parental (HuH-7) cells and cells with acquired resistance to sorafenib (HuH-7 R ). a Workflow for quantitative phosphoproteomic analyses between parental (HuH-7) and HCC cells with acquired sorafenib resistance (HuH-7 R ) via SILAC-based mass spectrometry. Heavy and light cell lysates were mixed and digested with trypsin and fractionated by high-pH reverse-phase chromatography. Phosphopeptides were then purified with TiO 2 column and analyzed in an LTQ-Orbitrap Velos hybrid mass spectrometer. b Clustered gene ontology functional enrichment was assessed with DAVID. Upregulated phosphoproteins in HuH-7 R cells showing a SILAC fold change H/L ≥ 1.5 were analyzed. The top three functional clusters are listed. −Log ( p values) and enrichment scores are presented. c Pathway enrichment analysis of the upregulated phosphoproteins in HuH-7 R cells based on the KEGG pathway database with DAVID. Pathways with p values

    Article Snippet: Liquid chromatography tandem MS analyses Enriched SILAC samples were analyzed separately with an LTQ-Orbitrap Velos mass analyzer (Thermo Fisher Scientific).

    Techniques: Mass Spectrometry, Reversed-phase Chromatography, Purification, Functional Assay