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PXD007145

PXD007145 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleBenchmarking LFQ, SILAC and MS2/MS3-based TMT quantification strategies for large-scale phosphoproteomics
DescriptionComprehensive 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.
HostingRepositoryPRIDE
AnnounceDate2018-03-13
AnnouncementXMLSubmission_2018-03-19_01:45:30.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterAlexander Hogrebe
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606; scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: 4932;
ModificationListTMT6plex-126 reporter+balance reagent acylated residue; phosphorylated residue; iodoacetamide derivatized residue
InstrumentOrbitrap Fusion Lumos
Dataset History
RevisionDatetimeStatusChangeLog 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
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
curator keyword: Technical
submitter keyword: phosphoproteomics, technical, benchmark, quantification, LFQ, SILAC, TMT, MS2, MS3
Contact List
Jesper V. Olsen
contact affiliationNovo Nordisk Foundation Center for Protein Research, Proteomics Program, University of Copenhagen, Denmark
contact emailjesper.olsen@cpr.ku.dk
lab head
Alexander Hogrebe
contact affiliationNNF Center for Protein Research, Copenhagen, Denmark
contact emailalexander.hogrebe@cpr.ku.dk
dataset submitter
Full Dataset Link List
Dataset FTP location
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