Dataset Information

Hypothesis-free Illustration on How Phosphorylation Dynamically Impact Protein Turnover

ABSTRACT: The turnover measurement of proteins and proteoforms has been largely facilitated by the metabolic labeling coupled with mass spectrometry (MS), such as the dynamic Stable isotope labeling by amino acids in cell culture (SILAC) experiments, i.e., the dynamic SILAC or pSILAC approach. Very recent studies including ours have integrated post-translational modification (PTM) proteomic methodology, such as phosphoproteomics, with pSILAC experiments in steady-state systems and revealed the link between protein PTM and turnover on the proteome-scale. In this study, we present a novel pSILAC phosphoproteomic dataset in a dynamic process of cell starvation using data-independent acquisition mass spectrometry (DIA-MS). We further propose a “hypothesis-free”, time series data analysis strategy to interrogate how phosphorylation dynamically impact protein turnover. With this strategy, we discovered complex relationship between phosphorylation and protein turnover that was previously underexplored. Our results further revealed potential biological features including phosphorylation stoichiometry that are associated with the peptideform turnover of phosphorylation. Our data also suggested that phosphoproteomic abundance regulation during starvation tends to be not correlated with turnover diversity, underscoring the importance of studying PTM site-resolved turnover datasets in the future.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Disease Free

SUBMITTER: Wenxue Li

LAB HEAD: Yansheng Liu

PROVIDER: PXD037360 | Pride | 2022-12-01

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20191129_pc12_NonAdh_pSIL_Ctr_12h_1.raw	Raw
	20191129_pc12_NonAdh_pSIL_Ctr_12h_2.raw	Raw
	20191129_pc12_NonAdh_pSIL_Ctr_12h_3.raw	Raw
	20191129_pc12_NonAdh_pSIL_Ctr_1h_1.raw	Raw
	20191129_pc12_NonAdh_pSIL_Ctr_1h_2.raw	Raw

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Publications

Toward a hypothesis-free understanding of how phosphorylation dynamically impacts protein turnover.

Li Wenxue W Salovska Barbora B Fornasiero Eugenio F EF Liu Yansheng Y

Proteomics 20221207 3-4

The turnover measurement of proteins and proteoforms has been largely facilitated by workflows coupling metabolic labeling with mass spectrometry (MS), including dynamic stable isotope labeling by amino acids in cell culture (dynamic SILAC) or pulsed SILAC (pSILAC). Very recent studies including ours have integrated themeasurement of post-translational modifications (PTMs) at the proteome level (i.e., phosphoproteomics) with pSILAC experiments in steady state systems, exploring the link between ...[more]

PMID: 36422574

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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