The Experts below are selected from a list of 3411 Experts worldwide ranked by ideXlab platform

Andreas Tholey - One of the best experts on this subject based on the ideXlab platform.

  • quantitative protease cleavage site profiling using Tandem Mass Tag labeling and lc maldi tof tof ms ms analysis
    Journal of Proteome Research, 2012
    Co-Authors: Thomas Jakoby, Bart H J Van Den Berg, Andreas Tholey
    Abstract:

    Knowledge of cleavage site specificity and activity are major prerequisites for understanding protease function. On the basis of a recently presented approach for proteomic identification of cleava...

  • Tandem Mass Tag Protein Labeling for Top-Down Identification and Quantification
    Analytical chemistry, 2011
    Co-Authors: Chien-wen Hung, Andreas Tholey
    Abstract:

    Top-down Mass spectrometry holds tremendous potential for characterization and quantification of intact proteins. So far, however, very few studies have combined top-down proteomics with protein quantification. In view of the success of isobaric Mass Tags in quantitative bottom-up proteomics, we applied the Tandem Mass Tag (TMT) technology to label intact proteins and examined the feasibility to directly quantify TMT-labeled proteins. A top-down platform encompassing separation via ion-pair reversed-phase liquid chromatography using monolithic stationary phases coupled online to an LTQ-Orbitrap Velos electron-transfer dissociation (ETD) Mass spectrometer (MS) was established to simultaneously identify and quantify TMT-labeled proteins. The TMT-labeled proteins were found to be readily dissociated under high-energy collision dissociation (HCD) activation. The liberated reporter ions delivered expected ratios over a wide dynamic range independent of the protein charge state. Furthermore, protein sequence ta...

Tianyu Zhang - One of the best experts on this subject based on the ideXlab platform.

  • Differential Protein Expression between Cystic and Solid Vestibular Schwannoma Using Tandem Mass Tag‐Based Quantitative Proteomic Analysis
    Proteomics. Clinical applications, 2020
    Co-Authors: Yuxuan Shi, Dongming Yin, Yang Zhang, Peidong Dai, Weidong Zhao, Tianyu Zhang
    Abstract:

    PURPOSE Cystic vestibular schwannoma (CVS) and solid vestibular schwannoma (SVS) are subgroups of vestibular schwannoma (VS). The tumorigenesis of CVS and SVS have not been fully elucidated, and this study is designed to identify differentially expressed proteins involved in the tumorigenesis of CVS and SVS. EXPERIMENTAL DESIGN Tandem Mass Tag-based proteomics is used to determine the protein expression profiles from CVS and SVS tissues. RESULTS A total of 30 differentially expressed proteins are identified between CVS and SVS, with 6 being upregulated and 24 being downregulated. Bioinformatics analyses are performed according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. These results indicate that two selected proteins (COL1A1 and COL1A2) are potential biomarkers for distinguishing CVS and SVS. CONCLUSIONS AND CLINICAL RELEVANCE Differentially expressed proteins linked to CVS and SVS are identified, and these proteins might provide potential biomarkers for human VS diagnosis. Furthermore, the present study supports the notion that decreased collagen might be the reason for bleeding associated with CVS.

  • differential protein expression between cystic and solid vestibular schwannoma using Tandem Mass Tag based quantitative proteomic analysis
    Proteomics Clinical Applications, 2020
    Co-Authors: Yuxuan Shi, Dongming Yin, Yang Zhang, Peidong Dai, Weidong Zhao, Tianyu Zhang
    Abstract:

    PURPOSE Cystic vestibular schwannoma (CVS) and solid vestibular schwannoma (SVS) are subgroups of vestibular schwannoma (VS). The tumorigenesis of CVS and SVS have not been fully elucidated, and this study is designed to identify differentially expressed proteins involved in the tumorigenesis of CVS and SVS. EXPERIMENTAL DESIGN Tandem Mass Tag-based proteomics is used to determine the protein expression profiles from CVS and SVS tissues. RESULTS A total of 30 differentially expressed proteins are identified between CVS and SVS, with 6 being upregulated and 24 being downregulated. Bioinformatics analyses are performed according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. These results indicate that two selected proteins (COL1A1 and COL1A2) are potential biomarkers for distinguishing CVS and SVS. CONCLUSIONS AND CLINICAL RELEVANCE Differentially expressed proteins linked to CVS and SVS are identified, and these proteins might provide potential biomarkers for human VS diagnosis. Furthermore, the present study supports the notion that decreased collagen might be the reason for bleeding associated with CVS.

Joao A Paulo - One of the best experts on this subject based on the ideXlab platform.

  • mTMT: An Alternative, Nonisobaric, Tandem Mass Tag Allowing for Precursor-Based Quantification
    Analytical chemistry, 2019
    Co-Authors: Joao A Paulo, Steven P. Gygi
    Abstract:

    Stable isotope labeling of peptides is the basis for numerous Mass-spectrometry-based quantification strategies. Isobaric Tagging and metabolic labeling, namely, Tandem Mass Tagging (TMT) and SILAC...

  • TKO6: A Peptide Standard To Assess Interference for Unit-Resolved Isobaric Labeling Platforms.
    Journal of proteome research, 2018
    Co-Authors: Joao A Paulo, José Navarrete-perea, Sanjukta Guha Thakurta, Steven P. Gygi
    Abstract:

    Protein abundance profiling using isobaric labeling is a well-established quantitative Mass spectrometry technique. However, ratio distortion resulting from coisolated and cofragmented ions, commonly referred to as interference, remains a drawback of this strategy. Tribrid Mass spectrometers, such as the Orbitrap Fusion and the Orbitrap Fusion Lumos with a triple Mass analyzer configuration, facilitate methods (namely, SPS-MS3) that can help alleviate interference. However, few standards are available to measure interference and thereby aid in method development. Here we introduce the TKO6 standard that assesses ion interference and is designed specifically for data acquired at low (unit) Mass resolution. We use TKO6 to compare interference in MS2- versus MS3-based quantitation methods, data acquisition methods of different lengths, and ion-trap-based Tandem Mass Tag reporter ion analysis (IT-MS3) with conventional Orbitrap-based analysis (OT-MS3). We show that the TKO6 standard is a valuable tool for assessing quantification accuracy in isobaric-Tag-based analyses.

  • Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3
    Journal of proteome research, 2018
    Co-Authors: José Navarrete-perea, Steven P. Gygi, Joao A Paulo
    Abstract:

    Mass spectrometry (MS) coupled toisobaric labeling has developed rapidly into a powerful strategy for high-throughput protein quantification. Sample multiplexing and exceptional sensitivity allow for the quantification of tens of thousands of peptides and, by inference, thousands of proteins from multiple samples in a single MS experiment. Accurate quantification demands a consistent and robust sample-preparation strategy. Here, we present a detailed workflow for SPS-MS3-based quantitative abundance profiling of Tandem Mass Tag (TMT)-labeled proteins and phosphopeptides that we have named the streamlined (SL)-TMT protocol. We describe a universally applicable strategy that requires minimal individual sample processing and permits the seamless addition of a phosphopeptide enrichment step ("mini-phos") with little deviation from the deep proteome analysis. To showcase our workflow, we profile the proteome of wild-type Saccharomyces cerevisiae yeast grown with either glucose or pyruvate as the carbon source. Here, we have established a streamlined TMT protocol that enables deep proteome and medium-scale phosphoproteome analysis.

  • an internal standard for assessing phosphopeptide recovery from metal ion oxide enrichment strategies
    Journal of the American Society for Mass Spectrometry, 2018
    Co-Authors: Joao A Paulo, Alison R Erickson, Jeffrey Knott, Jose Navarreteperea, Stephen P. Gygi
    Abstract:

    Phosphorylation-mediated signaling pathways have major implications in cellular regulation and disease. However, proteins with roles in these pathways are frequently less abundant and phosphorylation is often sub-stoichiometric. As such, the efficient enrichment, and subsequent recovery of phosphorylated peptides, is vital. Mass spectrometry-based proteomics is a well-established approach for quantifying thousands of phosphorylation events in a single experiment. We designed a peptide internal standard-based assay directed toward sample preparation strategies for Mass spectrometry analysis to understand better phosphopeptide recovery from enrichment strategies. We coupled Mass-differential Tandem Mass Tag (mTMT) reagents (specifically, TMTzero and TMTsuper-heavy), nine Mass spectrometry-amenable phosphopeptides (phos9), and peak area measurements from extracted ion chromatograms to determine phosphopeptide recovery. We showcase this mTMT/phos9 recovery assay by evaluating three phosphopeptide enrichment workflows. Our assay provides data on the recovery of phosphopeptides, which complement other metrics, namely the number of identified phosphopeptides and enrichment specificity. Our mTMT/phos9 assay is applicable to any enrichment protocol in a typical experimental workflow irrespective of sample origin or labeling strategy.

Steven P. Gygi - One of the best experts on this subject based on the ideXlab platform.

  • mTMT: An Alternative, Nonisobaric, Tandem Mass Tag Allowing for Precursor-Based Quantification
    Analytical chemistry, 2019
    Co-Authors: Joao A Paulo, Steven P. Gygi
    Abstract:

    Stable isotope labeling of peptides is the basis for numerous Mass-spectrometry-based quantification strategies. Isobaric Tagging and metabolic labeling, namely, Tandem Mass Tagging (TMT) and SILAC...

  • TKO6: A Peptide Standard To Assess Interference for Unit-Resolved Isobaric Labeling Platforms.
    Journal of proteome research, 2018
    Co-Authors: Joao A Paulo, José Navarrete-perea, Sanjukta Guha Thakurta, Steven P. Gygi
    Abstract:

    Protein abundance profiling using isobaric labeling is a well-established quantitative Mass spectrometry technique. However, ratio distortion resulting from coisolated and cofragmented ions, commonly referred to as interference, remains a drawback of this strategy. Tribrid Mass spectrometers, such as the Orbitrap Fusion and the Orbitrap Fusion Lumos with a triple Mass analyzer configuration, facilitate methods (namely, SPS-MS3) that can help alleviate interference. However, few standards are available to measure interference and thereby aid in method development. Here we introduce the TKO6 standard that assesses ion interference and is designed specifically for data acquired at low (unit) Mass resolution. We use TKO6 to compare interference in MS2- versus MS3-based quantitation methods, data acquisition methods of different lengths, and ion-trap-based Tandem Mass Tag reporter ion analysis (IT-MS3) with conventional Orbitrap-based analysis (OT-MS3). We show that the TKO6 standard is a valuable tool for assessing quantification accuracy in isobaric-Tag-based analyses.

  • Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3
    Journal of proteome research, 2018
    Co-Authors: José Navarrete-perea, Steven P. Gygi, Joao A Paulo
    Abstract:

    Mass spectrometry (MS) coupled toisobaric labeling has developed rapidly into a powerful strategy for high-throughput protein quantification. Sample multiplexing and exceptional sensitivity allow for the quantification of tens of thousands of peptides and, by inference, thousands of proteins from multiple samples in a single MS experiment. Accurate quantification demands a consistent and robust sample-preparation strategy. Here, we present a detailed workflow for SPS-MS3-based quantitative abundance profiling of Tandem Mass Tag (TMT)-labeled proteins and phosphopeptides that we have named the streamlined (SL)-TMT protocol. We describe a universally applicable strategy that requires minimal individual sample processing and permits the seamless addition of a phosphopeptide enrichment step ("mini-phos") with little deviation from the deep proteome analysis. To showcase our workflow, we profile the proteome of wild-type Saccharomyces cerevisiae yeast grown with either glucose or pyruvate as the carbon source. Here, we have established a streamlined TMT protocol that enables deep proteome and medium-scale phosphoproteome analysis.

Jesper V. Olsen - One of the best experts on this subject based on the ideXlab platform.

  • Limits for Resolving Isobaric Tandem Mass Tag Reporter Ions Using Phase-Constrained Spectrum Deconvolution
    Journal of proteome research, 2018
    Co-Authors: Christian D. Kelstrup, Konstantin Aizikov, Tanveer S. Batth, Arne Kreutzman, Dmitry Grinfeld, Oliver Lange, Daniel Mourad, Alexander Makarov, Jesper V. Olsen
    Abstract:

    A popular method for peptide quantification relies on isobaric labeling such as Tandem Mass Tags (TMT), which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a Tandem Mass spectrometer. However, with higher degrees of multiplexing, the smaller Mass differences between the reporter ions increase the Mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered Mass resolution on Orbitrap instruments. It is therefore important to determine the Mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here, we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da Mass differences using an ultra-high-field Orbitrap Mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions wit...

  • Limits for resolving Tandem Mass Tag reporter ions with identical integer Mass using phase constrained spectrum deconvolution
    2018
    Co-Authors: Christian D. Kelstrup, Konstantin Aizikov, Tanveer S. Batth, Arne Kreutzman, Dmitry Grinfeld, Oliver Lange, Daniel Mourad, Alexander Makarov, Jesper V. Olsen
    Abstract:

    A popular method for peptide quantification relies on isobaric labeling such as Tandem Mass Tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a Tandem Mass spectrometer. However, with higher degrees of multiplexing, the smaller Mass differences between the reporter ions increase the Mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered Mass resolution on Orbitrap instruments. It is therefore important to determine the Mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da Mass differences using an ultra-high-field Orbitrap Mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on TMT reporter ion quantification with identical integer Masses.