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DataSet Summary

  • HostingRepository: PRIDE
  • AnnounceDate: 2018-03-13
  • AnnouncementXML: Submission_2018-03-19_01:45:30.xml
  • DigitalObjectIdentifier:
  • ReviewLevel: Peer-reviewed dataset
  • DatasetOrigin: Original data
  • RepositorySupport: Unsupported dataset by repository
  • PrimarySubmitter: Alexander Hogrebe
  • Title: Benchmarking LFQ, SILAC and MS2/MS3-based TMT quantification strategies for large-scale phosphoproteomics
  • Description: Comprehensive mass spectrometry (MS)-based proteomics is now feasible, but reproducible and multiplexed quantification remains challenging especially for analysis of post-translational modifications (PTMs), such as phosphorylation. Here we compared the most popular quantification techniques for phosphoproteomics in context of cell-signaling studies: label-free quantification (LFQ), stable isotope labeling by amino acids in cell culture (SILAC) and MS2- and MS3-measured tandem mass tags (TMT). In a mixed species comparison with fixed phosphopeptide-ratios, we found LFQ and SILAC to be the most accurate techniques. MS2-based TMT suffered from substantial ratio compression, which MS3-based TMT could partly rescue. However, when analyzing phosphoproteome changes in the DNA damage response (DDR), we found that MS3-based TMT was outperformed by MS2-based TMT as it identified most significantly regulated phosphopeptides due to its higher precision and higher number of identifications. Finally, we show that the high accuracy of MS3-based TMT is crucial for determination of phosphorylation site stoichiometry using a novel multiplexing-dependent algorithm.
  • SpeciesList: scientific name: Homo sapiens (Human); NCBI TaxID: 9606; scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: 4932;
  • ModificationList: TMT6plex-126 reporter+balance reagent acylated residue; phosphorylated residue; iodoacetamide derivatized residue
  • Instrument: Orbitrap Fusion Lumos

Dataset History

VersionDatetimeStatusChangeLog Entry
02017-07-27 07:09:56ID requested
12018-03-13 01:50:09announced
22018-03-19 01:45:31announcedUpdated publication reference for PubMed record(s): 29535314.

Publication List

  1. Hogrebe A, von Stechow L, Bekker-Jensen DB, Weinert BT, Kelstrup CD, Olsen JV, Benchmarking common quantification strategies for large-scale phosphoproteomics. Nat Commun, 9(1):1045(2018) [pubmed]

Keyword List

  1. curator keyword: Technical
  2. submitter keyword: phosphoproteomics, technical, benchmark, quantification, LFQ, SILAC, TMT, MS2, MS3

Contact List

    Jesper V. Olsen
    • contact affiliation: Novo Nordisk Foundation Center for Protein Research, Proteomics Program, University of Copenhagen, Denmark
    • contact email: jesper.olsen@cpr.ku.dk
    • lab head:
    Alexander Hogrebe
    • contact affiliation: NNF Center for Protein Research, Copenhagen, Denmark
    • contact email: alexander.hogrebe@cpr.ku.dk
    • dataset submitter:

Full Dataset Link List

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  2. PRIDE project URI
Repository Record List

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