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.Because there is limited correlation between mRNA and protein levels/activity (1), 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).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). 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) or bead-based protein arrays (5) and mass cytometry (6) 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). 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 (8–10). Furthermore, shotgun mass spectrometry has been used to profile signaling pathways by enriching phosphorylated peptides through approaches such as antiphospho-tyrosine antibodies (11), extensive fractionation (12), or panels of antibodies to enrich for signaling nodes (13). Coupling isotopic labeling methods (14, 15) to MS allows relative quantification of detectable peptides between two or a small number of samples, but these methods do not provide the absolute abundances of the peptides detected, nor are they amenable to the analysis of large numbers of biological samples. For example, to achieve substantial depth of coverage, multidimensional biochemical fractionations are required (9), limiting the number of samples that can be analyzed. Relatively large sample consumption is a constraint for analyzing clinical specimens. Under-sampling remains an issue in data-dependent modes, and missing information in the data is substantial. Thus, untargeted mass spectrometry is capable of broad discovery, but does not have adequate throughput or reproducibility for more expansive biological or clinical studies.There has been tremendous growth in the application of targeted quantitative MS to quantify proteotypic peptides (16–19). In contrast to untargeted “shotgun” modes of MS, targeted MS focuses the full analytic capacity of the instrument on selected analytes of interest, moving from a stochastic sampling of the complex mixture where the instrument “decides” (nonreproducibly and with low precision) what is analyzed- to targeted, high-precision measurements of suites of proteins of interest that can be context-dependent based on the biological question being asked. The most widely used form of targeted MS is multiple reaction monitoring (MRM). MRM has been the clinical gold standard for decades in pharmaceutical research and in clinical reference laboratories for quantification of small molecules such as drug metabolites or metabolites that accumulate as a result of inborn errors of metabolism (20, 21). Although proteolysis of the biospecimen is a source of preanalytical variation for peptide quantification, careful selection of peptide analytes readily produces MRM-based assays of high precision (%CV ≤ 20%) (17, 22) that enable accurate quantification of reproducibly released tryptic peptides. Also, because MRM assays use internal standards (i.e. synthetic, stable isotope-labeled peptides that are spiked into the biospecimen), assays yield highly reproducible results when shared among laboratories and implemented on different instrument platforms (23), even at high multiplex levels on an international stage (22). The molecular specificity of MRM is very high, as it is conferred by three orthogonal physiochemical properties of each peptide: its mass, its retention properties on HPLC, and the production of a set of fragment ions of specific mass (of which three to five are usually monitored) detected at characteristic ratios. High specificity, coupled to the large linear range of MRM (>10³) and the ability to monitor analytes at scheduled times during the MRM run, render MRM assays highly multiplexable. A recent study shows scalability of MRM via analytical validation of four multiplex MRM assays (ranging from 156–169 plex) quantifying 645 human peptides (319 proteins) with median %CV<6 and successful reproduction of assay results internationally across three laboratories (22). Thus, MRM shows many advantages over alternative protein measurement technologies, including the capability to multiplex with ease, the use of internal standards (aiding reproducible quantification and cross-laboratory standardization), high specificity through direct measurement of the analyte, and relatively less time and cost associated with assay development.Although immuno-MRM has previously been applied to quantify unmodified protein abundances in body fluids (24, 25), cell lysates, and tissues (26), the feasibility and success rate for using immuno-MRM to quantify phosphopeptides and phospho-signaling has not been tested. In this study, we tested the possibility that MRM could be coupled to peptide immuno-affinity enrichment (27, 28) to enable multiplex quantification of phospho-signaling networks. As proof-of-concept, we developed a 69-plex immuno-MRM assay for quantifying phospho-signaling in the DNA damage response (DDR) network. The DDR is critical for maintaining genomic integrity, and mutations in the DDR are among the most frequently identified in tumors (29). In this study we show: (1) simultaneous analysis of modified and nonmodified isoforms of peptides, (2) multiplexed quantitative analysis of signaling events, and (3) applicability to a variety of sample types and conditions. The multiplexed assay presented in this study replaces 69 Western blots with a 40 min MRM-MS run, providing quantitative, precise, specific data, and establishing feasibility for developing targeted assay panels to many cell signaling networks. The ability to quantitatively measure a large array of phosphorylation events would have broad benefits for the biomedical research community, spanning fundamental biological studies through to PD characterizations of novel drug compounds and the development of companion diagnostics.