title: Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes.
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.
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Jennifer G
authors.initials: JG
authors.lastname: Abelin
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Jinal
authors.initials: J
authors.lastname: Patel
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Lu
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Caitlin M
authors.initials: CM
authors.lastname: Feeney
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Lola
authors.initials: L
authors.lastname: Fagbami
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Creech
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Roger
authors.initials: R
authors.lastname: Hu
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Daniel
authors.initials: D
authors.lastname: Lam
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Desiree
authors.initials: D
authors.lastname: Davison
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Lindsay
authors.initials: L
authors.lastname: Pino
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Jana W
authors.initials: JW
authors.lastname: Qiao
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Eric
authors.initials: E
authors.lastname: Kuhn
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Adam
authors.initials: A
authors.lastname: Officer
authors.affiliation: §Beth-Israel Deaconess Medical Center, Boston, Massachusetts, 02215;
authors.forename: Jianxue
authors.initials: J
authors.lastname: Li
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Susan
authors.initials: S
authors.lastname: Abbatiello
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Aravind
authors.initials: A
authors.lastname: Subramanian
authors.affiliation: §Beth-Israel Deaconess Medical Center, Boston, Massachusetts, 02215;
authors.forename: Richard
authors.initials: R
authors.lastname: Sidman
authors.affiliation: ¶Sanford-Burhnam Research Institute, La Jolla, California 92037.
authors.forename: Evan
authors.initials: E
authors.lastname: Snyder
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142;
authors.forename: Steven A
authors.initials: SA
authors.lastname: Carr
authors.affiliation: From the ‡Broad Institute of MIT and Harvard, 415 Main St. Cambridge, Massachusetts 02142; jjaffe@broadinstitute.org.
authors.forename: Jacob D
authors.initials: JD
authors.lastname: Jaffe
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D061986:MCF-7 Cells; D051379:Mice; D010750:Phosphoproteins / Q000032:analysis* / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D054852:Small Molecule Libraries / Q000494:pharmacology*
chemical_list: D010750:Phosphoproteins; D054852:Small Molecule Libraries