PXD004725-2

PXD004725 is an original dataset announced via ProteomeXchange.

Title	Time-resolved analysis of proteome dynamics by TMT-SILAC hyperplexing
Description	In 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.
HostingRepository	PRIDE
AnnounceDate	2017-10-24
AnnouncementXML	Submission_2017-10-24_02:55:58.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Tian Zhang
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	TMT6plex-126 reporter+balance reagent acylated residue; 6x(13)C labeled residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2016-08-08 05:52:34	ID requested
1	2016-12-20 19:00:50	announced
⏵ 2	2017-10-24 02:55:59	announced	Updated project metadata.
3	2024-10-22 04:28:46	announced	2024-10-22: Updated project metadata.

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]

submitter keyword: proteome dynamics, TMT, SILAC, TMT-SILAC, hyperplexing, protein degrdation, protein synthesis,human primary fibroblasts

Sina Ghaemmaghami
contact affiliation	Biology Department, University of Rochester
contact email	sghaemma@bio.rochester.edu
lab head
Tian Zhang
contact affiliation	UNIVERSITY OF ROCHESTER
contact email	tzhang24@ur.rochester.edu
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD004725

PRIDE project URI

• PRIDE
  1. PXD004725
     1. Label: PRIDE project
     2. Name: Time-resolved analysis of proteome dynamics by TMT-SILAC hyperplexing