Dataset Information

Time-resolved analysis of proteome dynamics by TMT-SILAC hyperplexing

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Fibroblast

SUBMITTER: Tian Zhang

LAB HEAD: Sina Ghaemmaghami

PROVIDER: PXD004725 | Pride | 2016-12-14

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	SILAClabeledSina_SILACRatios-HeavySearch_16_015.csv	Csv
	SILACunlabeledSina_SILACRatios_16-015.csv	Csv
	Sina_12hours-TMT127C_16-015-SILACHeavyLabeledSearch.msf	Msf
	Sina_12hours-TMT127C_16-015.msf	Msf
	Sina_12hours-TMT127C_16-015.raw	Raw

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Publications

Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing.

Welle Kevin A KA Zhang Tian T Hryhorenko Jennifer R JR Shen Shichen S Qu Jun J Ghaemmaghami Sina S

Molecular & cellular proteomics : MCP 20161020 12

Recent advances in mass spectrometry have enabled system-wide analyses of protein turnover. By globally quantifying the kinetics of protein clearance and synthesis, these methodologies can provide important insights into the regulation of the proteome under varying cellular and environmental conditions. To facilitate such analyses, we have employed a methodology that combines metabolic isotopic labeling (Stable Isotope Labeling in Cell Culture - SILAC) with isobaric tagging (Tandem Mass Tags - T ...[more]

PMID: 27765818

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Project 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.

Project description:To ensure that the pool of precursor amino acids utilized for protein synthesis is completely labeled prior to the first labeling time-point, we conducted an isotopomer analysis. After adaption to media, cells were plated at a density of 500,000 cells per 10 cm plate. 8 days after plating, the confluent quiescent cultures were switched to 15N labeling medium. Cells were collected after 0h, 6h, 1d, 2d, 4d of labeling, washed with PBS and cell pellets were frozen prior to further analysis. 15N labeling medium was made from “Cell free” Amino Acid Mix (20AA, U-15N, 96-98%)(Cambridge Isotope Laboratories) and supplemented with 15% dialyzed fetal bovine serum (Thermo Scientific), 100U/mL penicillin, 100U/mL streptomycin. 1g is used for making 702mL labeling medium. The final amino acids concentrations in 15N labeling medium are as following: Alanine: 0.1140 g/L, Arginine: 0.0499 g/L, Asparagine: 0.0940 g/L, Aspartic acid: 0.1353 g/L, Cystine: 0.01140 g/L, Glutamic acid: 0.1852 g/L, Glutamine: 0.0869 g/L, Glycine: 0.0698 g/L, Histidine: 0.0157 g/L, Isoleucine: 0.0698 g/L, Leucine: 0.1211 g/L, Lysine: 0.0527 g/L, Methionine: 0.0228 g/L, Phenylalanine: 0.0541 g/L, Proline: 0.0299 g/L, Serine: 0.0541 g/L, Threonine: 0.0741 g/L, Tryptophan: 0.0456 g/L, Tyrosine: 0.0613g/L, Valine: 0.0798 g/L. LC-MS/MS and quantitative analysis of isotopic envelopes and 15N incorporation of peptides was conducted as described in (Zhang et al., 2014). The data indicate that when exposed to fully 15N-labeled media (where all 20 natural amino acids are isotopically labeled), the fraction of proteins that are synthesized within the first day are almost fully labeled. Thus, the extent of fractional labeling is almost exclusively influenced by the kinetics of protein turnover rather than amino acid uptake and recycling within the cell. Table of files Cells Time points Labeling Fractionation Raw Data Filenames Wildtype 0d, 1d,2d,4d,7d13C 6Lys, 6Arg& 15N No 0d.raw 1d.raw 2d.raw 4d.raw 7d.raw