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Mol Cell Proteomics DOI:10.1074/mcp.M116.058354

Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes.

Publication TypeJournal Article
Year of Publication2016
AuthorsAbelin, JG, Patel, J, Lu, X, Feeney, CM, Fagbami, L, Creech, AL, Hu, R, Lam, D, Davison, D, Pino, L, Qiao, JW, Kuhn, E, Officer, A, Li, J, Abbatiello, S, Subramanian, A, Sidman, R, Snyder, E, Carr, SA, Jaffe, JD
JournalMol Cell Proteomics
Volume15
Issue5
Pages1622-41
Date Published2016 May
ISSN1535-9484
Abstract

Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens.To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed ∼95% of the reduced-representation phosphopeptide probes to be detected in ∼200 samples.The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures.

URLhttp://www.mcponline.org/cgi/pmidlookup?view=long&pmid=26912667
DOI10.1074/mcp.M116.058354
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/26912667?dopt=Abstract

Alternate JournalMol. Cell Proteomics
PubMed ID26912667
PubMed Central IDPMC4858944
Grant ListU01 CA164186 / CA / NCI NIH HHS / United States
U24 CA160034 / CA / NCI NIH HHS / United States
U54 HG008097 / HG / NHGRI NIH HHS / United States