Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling

In most cell signaling experiments, analytes are measured one Western blot lane at a time in a semiquantitative and often poorly specific manner, limiting our understanding of network biology and hindering the translation of novel therapeutics and diagnostics. We show the feasibility of using multiplex immuno-MRM for phospho-pharmacodynamic measurements, establishing the potential for rapid and precise quantification of cell signaling networks. A 69-plex immuno-MRM assay targeting the DNA damage response network was developed and characterized by response curves and determinations of intra- and inter-assay repeatability. The linear range was ≥3 orders of magnitude, the median limit of quantification was 2.0 fmol/mg, the median intra-assay variability was 10% CV, and the median interassay variability was 16% CV. The assay was applied in proof-of-concept studies to immortalized and primary human cells and surgically excised cancer tissues to quantify exposure–response relationships and the effects of a genomic variant (ATM kinase mutation) or pharmacologic (kinase) inhibitor. The study shows the utility of multiplex immuno-MRM for simultaneous quantification of phosphorylated and nonmodified peptides, showing feasibility for development of targeted assay panels to cell signaling networks. In most cell signaling experiments, analytes are measured one Western blot lane at a time in a semiquantitative and often poorly specific manner, limiting our understanding of network biology and hindering the translation of novel therapeutics and diagnostics. We show the feasibility of using multiplex immuno-MRM for phospho-pharmacodynamic measurements, establishing the potential for rapid and precise quantification of cell signaling networks. A 69-plex immuno-MRM assay targeting the DNA damage response network was developed and characterized by response curves and determinations of intra- and inter-assay repeatability. The linear range was ≥3 orders of magnitude, the median limit of quantification was 2.0 fmol/mg, the median intra-assay variability was 10% CV, and the median interassay variability was 16% CV. The assay was applied in proof-of-concept studies to immortalized and primary human cells and surgically excised cancer tissues to quantify exposure–response relationships and the effects of a genomic variant (ATM kinase mutation) or pharmacologic (kinase) inhibitor. The study shows the utility of multiplex immuno-MRM for simultaneous quantification of phosphorylated and nonmodified peptides, showing feasibility for development of targeted assay panels to cell signaling networks. Because there is limited correlation between mRNA and protein levels/activity (1.Ghazalpour A. Bennett B. Petyuk V.A. Orozco L. Hagopian R. Mungrue I.N. Farber C.R. Sinsheimer J. Kang H.M. Furlotte N. Park C.C. Wen P.-Z. Brewer H. Weitz K. Camp D.G. Pan C. Yordanova R. Neuhaus I. Tilford C. Siemers N. Gargalovic P. Eskin E. Kirchgessner T. Smith D.J. Smith R.D. Lusis A.J. Comparative analysis of proteome and transcriptome variation in mouse.PLoS Genet. 2011; 7: e1001393Crossref PubMed Scopus (428) Google Scholar), quantification of proteins and post-translational modifications is critical to understanding cellular signaling and determining pharmacodynamic (PD) 1 responses. Phosphorylation is a key post-translational modification used in signaling networks to modulate protein/pathway activity, protein interactions, and protein localization in response to extracellular and intracellular stimuli. Many diseases exhibit dysfunctions in signaling networks, and thus major efforts to identify novel drug targets (e.g. kinase inhibitors) are based on signal transduction pathways (2.Persidis A. Signal transduction as a drug-discovery platform.Nat. Biotechnol. 1998; 16: 1082-1083Crossref PubMed Scopus (45) Google Scholar). Currently the research community lacks high throughput, quantitative tools for studying phospho-signaling networks, hindering our basic understanding of network biology and hence the translation of novel therapeutics and companion diagnostics. In most experiments, one analyte is measured one Western blot lane at a time in a semiquantitative and often nonspecific manner. These drawbacks limit our ability to extend knowledge beyond individual phosphorylation events to a system-wide study of phosphorylation dynamics, which is critical because signal transduction pathways act as interconnected networks, and the effects of mutations in individual genes (as well as the effects of pharmacologic compounds) spread throughout the network (3.Barabási A.-L. Network medicine – from obesity to the “diseasome.”.N. Engl. J. Med. 2007; 357: 404-407Crossref PubMed Scopus (403) Google Scholar). Although Western blotting and related traditional immuno-assay platforms (e.g. ELISA) have been pushed brilliantly to their limits and have formed the basis of many advances in biomedical research, they are inadequate to support the needs of the postgenomic world, in which we need innovative technologies for determining the effects of any experimental condition (e.g. agonist or antagonist exposures, genetic variations) on the major signal transduction networks of the human cell, using precise, standardized, moderate-to-high throughput methods that can be reproduced across laboratories. Newer technologies, such as planar (4.Akbani R. Becker K.-F. Carragher N. Goldstein T. de Koning L. Korf U. Liotta L. Mills G.B. Nishizuka S.S. Pawlak M. Petricoin E.F. Pollard H.B. Serrels B. Zhu J. Realizing the promise of reverse phase protein arrays for clinical, translational, and basic research: a workshop report: the RPPA (Reverse Phase Protein Array) society.Mol. Cell. Proteomics. 2014; 13: 1625-1643Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) or bead-based protein arrays (5.Houser B. Bio-Rad's Bio-Plex® suspension array system, xMAP technology overview.Arch. Physiol. Biochem. 2012; 118: 192-196Crossref PubMed Scopus (147) Google Scholar) and mass cytometry (6.Liu R. Wu P. Yang L. Hou X. Lv Y. Inductively coupled plasma mass spectrometry-based immunoassay: a review.Mass Spectrom. Rev. 2014; 33: 373-393Crossref PubMed Scopus (84) Google Scholar) have shown potential for improving our ability to quantify signaling networks. However, like traditional immunoassays, these techniques do not directly detect and quantify the target analyte. Rather, the concentration of the target is inferred from a reporter signal, such as a fluorescent or mass tag on the antibody. As a result, these assay platforms are plagued by interferences present in biological matrices, which undermine the specificity of the assays in all but the most rigorously optimized settings using highly monospecific antibodies (7.Hoofnagle A.N. Wener M.H. The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry.J. Immunol. Methods. 2009; 347: 3-11Crossref PubMed Scopus (381) Google Scholar). Thus, generating such assays and assuring specificity is costly, time-consuming, and very difficult, especially in multiplex. Technological advancements in MS have enabled an impressive depth of coverage of the phosphoproteome using untargeted (“shotgun”) approaches (8.Ellis M.J. Gillette M. Carr S.A. Paulovich A.G. Smith R.D. Rodland K.K. Townsend R.R. Kinsinger C. M. H. genomic to cancer biology the PubMed Scopus Google P. Yang T. 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