Dataset Information

Phosphoproteomic profiling of the DNA damage response in MCF10A and human PBMC cells by SILAC labeling and 1D and 2D-LC-MS/MS

ABSTRACT: The DNA-damage response (DDR) is a critical network for maintaining genomic integrity, and mutations in the DDR are among the most frequently identified in tumors. Phosphorylation is a key signaling event in the DDR network. We sought to design high throughput, mass spectrometry based assays to monitor phosphopeptides in the DDR network for biological experiments and pharmacodynamics studies. These assays require an enrichment step, which can be done using antibodies. Developing antibodies de novo for phosphopeptide enrichment is costly and time-consuming, so we assessed the feasibility of immobilized-metal affinity chromatography (IMAC) enrichment coupled with multiple reaction monitoring-mass spectrometry (MRM-MS) for precise measurement of large numbers of phosphopeptides in the DDR network. Towards this goal, we evaluate the ability of the approach to reproducibly measure the endogenous phosphorylation levels of proteins associated with the DDR. Reproducibly detectable phosphopeptide targets for MRM-based assay development were empirically identified from LC-MS/MS analyses of SILAC-labeled MCF10A human breast epithelial cells exposed or mock-exposed to DNA damage. Samples were treated in triplicate with either ionizing radiation (IR) or methyl methanesulfonate (MMS) as the DNA-damaging agent. SILAC-labeled cells were prepared in two separate (label swap) experiments. First, treated cell lines were grown in heavy SILAC medium and untreated in light, and second, this labeling scheme was reversed. Untreated and treated cells were mixed at a 1:1 ratio by protein mass. To identify phosphopeptides at levels observable by MRM in an approach amenable to moderate throughput, a single step IMAC enrichment was developed for rapid processing of whole cell lysate.

INSTRUMENT(S): LTQ Orbitrap Velos, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Epithelial Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Jacob Kennedy

LAB HEAD: Amanda G Paulovich

PROVIDER: PXD002363 | Pride | 2015-12-01

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	MCF10A_PO4_SILAC_Heavy_IR_1D_Rep1A.raw	Raw
	MCF10A_PO4_SILAC_Heavy_IR_1D_Rep1B.raw	Raw
	MCF10A_PO4_SILAC_Heavy_IR_1D_Rep2A.raw	Raw
	MCF10A_PO4_SILAC_Heavy_IR_1D_Rep2B.raw	Raw
	MCF10A_PO4_SILAC_Heavy_IR_1D_Rep3A.raw	Raw

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Publications

Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling.

Kennedy Jacob J JJ Yan Ping P Zhao Lei L Ivey Richard G RG Voytovich Uliana J UJ Moore Heather D HD Lin Chenwei C Pogosova-Agadjanyan Era L EL Stirewalt Derek L DL Reding Kerryn W KW Whiteaker Jeffrey R JR Paulovich Amanda G AG

Molecular & cellular proteomics : MCP 20151130 2

A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-base ...[more]

PMID: 26621847

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Project description:Wild type yeast cells were metabolically labeled in SILAC medium. The heavy (H)-SILAC-labeled cells were then continuously exposed to 0.01% MMS to induce replication stress, and the light (L)-SILAC-labeled cells were mock-exposed. After 3 hours, heavy and light cells were harvested and lysed. To ensure reproducibility, the entire experiment was repeated, and the labels were swapped such that the (L)-SILAC-labeled wild type yeast cells were exposed to 0.01% MMS for 3 hours, and the (H)-SILAC-labeled wild type yeast cells were mock-exposed. Lysates from heavy and light cells were mixed 1:1 by protein mass, reduced and treated with iodoacetamide and then digested with trypsin. 5 mg of each protein lysate was fractionated by high-pH reverse phase (RP) liquid chromatography into 12 samples. For proteome analysis, 2% of each fraction was dried down and re-suspended for LC-MS/MS analysis. The remaining 98% was processed to enrich for phosphopeptides using immobilized metal affinity chromatography (IMAC), dried down, and re-suspended in 0.1% FA, 3% ACN for LC-MS/MS analysis. Global and phosphopeptide-enriched samples were analyzed by LC-MS/MS on a Thermo LTQ-Orbitrap Velos mass spectrometer. Raw MS/MS spectra were searched against version 3.69 of the Yeast International Protein Index (IPI) sequence database using three independent search engines (MaxQuant/Andromeda, Spectrum Mill, and xTandem). All searches were performed with the tryptic enzyme constraint set for up to two missed cleavages, oxidized methionine set as a variable modification, and carbamidomethylated cysteine set as a static modification. For MaxQuant, the peptide MH+ mass tolerances were set at 20 ppm. For X!Tandem, the peptide MH+ mass tolerances were set at ±2.0 Da with post-search filtering of the precursor mass to 50 ppm and the fragment MH+ mass tolerances were set at ±0.5 Da. For Spectrum Mill, peptide MH+ mass tolerances were set at 20 ppm and fragment MH+ mass tolerances were set at ±0.7 Da. The overall FDR was set at ≤3% based on a decoy database search. Phosphosite localization probabilities are reported in the MaxQuant results. Any site with a probability greater than 0.8 was considered to be localized. Quantification of the Heavy:Light ratios was performed using MaxQuant software, with a minimum ratio count of 2 and using unique + razor peptides for quantification.