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PXD004725

PXD004725 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleTime-resolved analysis of proteome dynamics by TMT-SILAC hyperplexing
DescriptionIn this project, a more efficient methodology for obtaining multi-timepoint kinetic data for proteome-wide analyses of protein turnover is developed and used to globally measure the kinetics of protein turnover in primary human fibroblasts (HCA2-hTert). This approach takes advantage of the multiplexing capabilities of isobaric tandem mass tags (TMT) to measure fractional SILAC labeling of multiple time-points in a single LC-MS/MS run. Human dermal fibroblasts (HCA2-hTert) (19, 20) were maintained in Eagle’s Minimum Essential Medium (ATCC) supplemented with 15% fetal bovine serum (Invitrogen), 100 U/mL penicillin, 100 U/mL streptomycin at 37℃ with 5% CO2. The media utilized for isotopic labeling was Eagle’s minimum essential medium (ATCC) supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were gradually adapted to the labeling media and were then plated at a density of 500,000 cells per 10 cm plate. For dividing cells, one day after plating, the cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl (13C6, 99%) and L-lysine:2HCl (13C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/l and 0.087 g/l and 15% dialyzed fetal bovine serum (Thermo Scientific) and were subsequently collected after 0, 24, 48, 72 hours of labeling. For quiescent cells, the cultures were grown for 8 days to achieve a state of quiescence by contact inhibition. The confluent quiescent cultures were switched to the labeling media and cells were collected after 0, 6, 12, 24, 36, 48, 72, 96, 144, 192, 336 hours of labeling. All cells were washed with PBS and frozen as cell pellets prior to further analysis.
HostingRepositoryPRIDE
AnnounceDate2024-10-22
AnnouncementXMLSubmission_2024-10-22_04:28:40.204.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterTian Zhang
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListTMT6plex-126 reporter+balance reagent acylated residue; 6x(13)C labeled residue; monohydroxylated residue; iodoacetamide derivatized residue
InstrumentQ Exactive
Dataset History
RevisionDatetimeStatusChangeLog Entry
02016-08-08 05:52:34ID requested
12016-12-20 19:00:50announced
22017-10-24 02:55:59announcedUpdated project metadata.
32024-10-22 04:28:46announced2024-10-22: Updated project metadata.
Publication List
Welle KA, Zhang T, Hryhorenko JR, Shen S, Qu J, Ghaemmaghami S, Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing. Mol Cell Proteomics, 15(12):3551-3563(2016) [pubmed]
Keyword List
submitter keyword: TMT-SILAC, protein degrdation, TMT,proteome dynamics, SILAC,human primary fibroblasts, protein synthesis, hyperplexing
Contact List
Sina Ghaemmaghami
contact affiliationBiology Department, University of Rochester
contact emailsghaemma@bio.rochester.edu
lab head
Tian Zhang
contact affiliationUNIVERSITY OF ROCHESTER
contact emailtzhang24@ur.rochester.edu
dataset submitter
Full Dataset Link List
Dataset FTP location
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PRIDE project URI
Repository Record List
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