Abstract SY44-04: Targeted MRM mass spectrometry enables highly multiplexed quantification of cell signaling in response to DNA damage
Authors: Paulovich Amanda G. 
Published: August 2015

The greatest challenge created by modern genomics is to understand how genomic variations contribute to disease phenotypes and to exploit this knowledge to develop novel therapeutics and diagnostics for personalized medicine. Proteins are obligatory intermediaries between genomic profiles and clinical/biological phenotypes. Proteins are required for virtually all biological processes, and especially for information processing by signaling networks, including receptors, kinases, and substrates. Because most diseases exhibit dysfunctions in cellular signaling networks that transduce external signals (e.g. hormones, neurotransmitters, cytokines) into cellular responses, most efforts identifying novel drug targets are based on knowledge of signal transduction pathways and their links to disease. Because there is limited correlation between mRNA, copy number variations, and protein levels/activity, direct quantification of proteins and posttranslational modifications is critical to understanding cellular signaling and elucidating genotype-phenotype relationships. Thus, although genomics is playing a critical role in personalized medicine, whetting our appetite for such advances, it is only a partial solution, and proteomics remains a necessary yet missing link between the genome and clinical phenotypes.

Remarkably, over a decade since the completion of the human genome sequence, the majority of human proteins cannot be measured, and the research community is reliant on antiquated protein detection methods (e.g. Western blotting, immunohistochemistry) that are semi-quantitative, irreproducible, and often non-specific. Indeed, a lack of rigorously validated reagents and a reliance on a small number of semi-quantitative results from Western blotting have been explicitly cited as one source of the irreproducibility of preclinical research. Additionally, the lack of standardized tools for reliably quantifying human proteins renders most of the exciting leads coming from ‘omics experiments not actionable and renders the human proteome clinically inaccessible. The cost is extraordinary, since the market for protein-detection antibodies is $2 billion/year, and it is well documented that over half do not work as claimed.

A rapidly growing trend in protein quantification is a targeted mass spectrometry (MS)-based method called Multiple Reaction Monitoring (MRM). MRM-based assays are highly specific, multiplexable, and precise. Because MRM-based assays can be standardized, reproduced, and thus shared across laboratories and instrument platforms, the MRM technology can circumvent roadblocks to the reproducibility of preclinical research. MRM-MS has been used for decades in pharmaceutical research and clinical reference laboratories for quantification of small molecules such as drug metabolites. As such, MRM has already had to pass muster with the Food & Drug Administration, paving the way for proteomic applications.

Data will be presented demonstrating that multiplex MRM assays can be used to obtain pharmacodynamic profiles of human cells responding to genotoxic stress, demonstrating feasibility of developing a multiplex MRM-based tool for assessing the activity of all core cell signaling pathways (a rapid “cell diagnostic test”). The tool could be standardized to support high inter-laboratory reproducibility, addressing recent widely publicized concerns about the reliability of preclinical research, and would provide a signaling network-based tool for screening compounds for off-target effects, drug repurposing, identifying complementary compounds for combination therapies, determining mechanisms of resistance, and identifying companion diagnostics. We know that cell signaling acts through interconnected networks that are dynamic and rapidly re-wire in response to perturbations; semi-quantitative, single-analyte approaches (e.g. Western blotting) offer a limited look at critical network biology. MRM provides a multiplex platform that can replace singular approaches with a method to reliably and precisely quantify the activity state of networks, going beyond single markers. Investigators need no longer be stuck looking under the lamppost for predicted phenotypes. Instead, they can cast the phenotypic net broadly, and let the biology declare itself. MRM can change the way people think about their experiments; in the same way as microarrays stimulated network biology at the gene expression level, MRM can do so at the cell signaling level.

To capitalize on the ability of MRM to standardize protein measurements, the ability to run MRM-based assays must be made readily available to the research community in the form of validated assays that can be run in individual laboratories or readily implemented in proteomic core facilities. Towards this end, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating standard operating protocols, reagents, and assay characterization data for highly validated assays. Additionally, a recent workshop co-sponsored by NCI and NHLBI established fit-for-purpose assay validation guidelines for MRM-based assays to maintain the high caliber of targeted protein assays.

Citation Format: Amanda G. Paulovich. Targeted MRM mass spectrometry enables highly multiplexed quantification of cell signaling in response to DNA damage. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY44-04. doi:10.1158/1538-7445.AM2015-SY44-04