PXD004937-1

PXD004937 is an original dataset announced via ProteomeXchange.

Title	Global up-regulation of protein degradation in quiescent cells
Description	In this project, we employed a dynamic mass spectrometry-based proteomic approach to obtain global maps of basal autophagic flux in human primary fibroblasts (HCA2-hTert). The data provide a comparison of protein degradation rates between dividing and quiescent HCA2-hTert cells. To further investigate the role of autophagy in up-regulated protein degradation, protein degradation rates were also measured in autophagy-deficient (Atg5-/-) in both dividing and quiescent cells. Steady-state protein level changes in dividing and quiescent cells were measured by standard SILAC. To investigate the role of PSME1 in proteasome activity regulation, protein degradation rates in PSME1 knockdown cell line were measured by SILAC. Stable isotope labeling 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. Both wildtype and Atg5-/- Cells were gradually adapted to this media and prepared for labeling experiments. Cells were then plated at a density of 500,000 cells per 10 cm plate. One day after plating, the dividing 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). Cells were collected after 0, 1, 2, and 3 days of labeling, washed with PBS and cell pellets were frozen prior to further analysis. 8 days after plating, the confluent quiescent 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). Cells were collected after 0, 2, 4, and 6 days of labeling, washed with PBS and cell pellets were frozen prior to further analysis. In order to measure changes in steady-state protein levels by standard SILAC, WT cells were gradually adapted to Eagle’s minimum essential medium (ATCC) supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin. Then, WT cells were passaged in MEM labeling media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl (13 C6, 99%) and L-lysine:2HCl (13 C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/l and 0.087 g/l and 15% dialyzed fetal bovine serum (Thermo scientific) for eight passages. Cells were then plated at a density of 500,000 cells per 10-cm plate. 2 days after plating, dividing cells were collected and washed with PBS, and cell pellets were frozen prior to further analysis. Following extraction (see below), equal protein amounts of dividing and quiescent WT cells were mixed before mass spectrometric analysis. To measure protein degradation in PSME1 knockdown cell line, 8 days after plating, the confluent quiescent cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with L-arginine:HCl (13C6, 99%) and L-lysine:2HCl (13D4, 96%-98%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/l and 0.087 g/l and 15% dialyzed fetal bovine serum (Thermo Scientific). Cells were collected after 3 days of labeling, washed with PBS and cell pellets were frozen prior to further analysis.
HostingRepository	PRIDE
AnnounceDate	2017-11-01
AnnouncementXML	Submission_2017-11-01_05:04:22.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	6x(13)C labeled residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2016-09-12 03:39:05	ID requested
⏵ 1	2017-11-01 05:04:23	announced

Dataset with its publication pending

curator keyword: Biological

submitter keyword: Human primary cells, protein degradation rates, dynamic SILAC, Dividing, Quiescence, SILAC, autophagy, proteasome, PSME1

Sina Ghaemmaghami
contact affiliation	Department of Biology, 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

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PRIDE project URI

• PRIDE
  1. PXD004937
     1. Label: PRIDE project
     2. Name: Global up-regulation of protein degradation in quiescent cells