Proteomics

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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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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]

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