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

Abstract

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 interassay 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 ( Because there is limited correlation between mRNA and protein levels/activity (1), quantification of proteins and posttranslational 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 tec...

Peptide Immunoaffinity Enrichment with Targeted Mass Spectrometry: Application to Quantification of ATM Kinase Phospho-Signaling

“…We previously demonstrated the application of a multiplexed immuno-MRM assay targeting phospho-signaling in response to DNA damage [10]. The assay measured the response of 69 analytes (phosphorylated and non-modified peptides) to demonstrate: i) multiplexed quantitative analysis of signaling events, ii) simultaneous analysis of modified and non-modified isoforms of peptides, and iii) applicability to a variety of sample types and conditions.…”

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“…Immuno-SRM has been successfully implemented to address the detection-limit challenges associated with measuring low-abundance protein biomarkers in the low- and sub-μg/L range in a wide array of studies. 48–51,58–62 …”

2017 White Paper on Recent Issues in Bioanalysis: Rise of Hybrid LBA/LCMS Immunogenicity Assays (Part 2: Hybrid LBA/LCMS Biotherapeutics, Biomarkers & Immunogenicity Assays and Regulatory Agencies’ Inputs)

“…While comparing current industry standards versus previous White Paper recommendations [8,11,14], there was consensus that one of the methodologies that are proving to be invaluable for tissue-based measurement is extraction of proteins using a protein precipitation step with high organic solvent, pellet digestion followed by the use of antipeptide antibodies for enrichment of tryptic peptides and subsequent measurement by LCMS [23][24][25]. Even though custom reagent generation is required, this technique enables the use of harsh extraction conditions including strong detergents for maximized recovery of proteins that may otherwise be insoluble, for example, membrane/structural proteins.…”