Multi-dimensional targeted mass spectrometry technology for pathway-scale functional proteomics

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Molecular & Cellular Analysis Technologies
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Not Applicable
Project SummaryThe advent of molecular biology and molecular profiling in clinical medicine has transformed our understandingof the molecular basis of human cancer. As a result, we are increasingly improving the classification of humantumors based on their specific genetic and molecular mechanisms of pathogenesis. However, currently only asmall number of mutant alleles guide treatment decisions, while most observed mutations remain of unknownpathologic and clinical significance. In addition, even for recently approved drugs, such as those targetingactivated kinase signaling, clinical efficacy is highly varied, with no currently satisfactory means to identifymolecular markers of response and resistance. Quantitative measurements of the abundance of proteins andstoichiometry of their regulatory post-translational modifications can be used to determine activation states ofof pathways and cells. However, current quantitative mass spectrometry techniques are limited by peptide ionfragmentation, duty cycles that restrict assays to about 100 proteins, and limited scalability to permit high-throughput clinical applications. To address this need, and broadly enable transformative future advances inprecision oncology and patient outcomes, we have recently developed a new method with 3 orders ofmagnitude improvement in sensitivity, termed accumulated ion monitoring (AIM). Using AIM, we developedthe Quantitative Cancer Proteomics Atlas (QCPA) for functional profiling of biochemical processes mediatingaberrant survival of cancer cells. In principle, this technology permits highly multiplexed, quantitative analysisof the expression and biochemical activity of thousands of proteins, covering most recurrently mutated andknown pathogenic pathways in cancer cells, and designed to be applied to clinically-accessible, microgrampatient specimens. The objective of this proposal is to develop scalable and high-throughput massspectrometry technology for proteome-wide and pathway-scale profiling of hundreds of clinical specimens onthe hours time scale. Our central hypothesis is that implementation of high-efficiency duty-cycle and ionmultiplexing quantitative proteomics will enable high-throughput functional molecular profiling for both basicscience and clinical applications. Aim 1 will develop pathway-scale functional mass spectrometry proteomicmapping technology based on multiplex and triggered ion monitoring. Aim 2 will implement tandem masstagging for high-throughput quantitative mass spectrometry for scalable functional profiling of clinical cancerspecimens. Successful completion of this proposal is expected to close the technical gap currently preventingthe use of mass spectrometry for comprehensive functional profiling of clinical specimens. This research willhave broad significance because improved quantitative functional measures of cell signaling are needed toovercome persistent challenges that limit progress in cancer biology and clinical oncology.