Quantifying post-translational modifications and protein expression by HTP-MS

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DESCRIPTION (provided by applicant): This application responds to the needs and opportunities in Applying Genomics and Other High Throughput Technologies. We aim to use a newly-developed mass spectroscopy technique, FLEXIQuant, to provide absolute quantitation of the levels of 1,000 proteins in several different cell lines. At the same time as providing quantitation, FLEXIQuant provides information on which peptides in a protein are modified, and to what extent. Our ultimate deliverables from this project will be as follows: (1) A set of 1,000 clones in FLEXIQuant vectors, deposited with a non-profit plasmid repository and freely available to the community; (2) for the proteins corresponding to these clones, a comprehensive database of peptides detectable in mass spectroscopy experiments after digestion with 4 different proteases; (3) for the mRNAs corresponding to these clones, a comprehensive database quantitating the levels of mRNAs in 6 cell lines under different conditions; and (4) a database comparing mRNA levels, protein levels, and post- translational modifications for these clones in the same cell lines, under the same conditions. This work will for the first time open up the study of post-translational modifications on the genomic level, and will provide a range of important data for analysis by the community. Types of questions/approaches this dataset will allow include (1) monitoring the level of and modifications on any of the proteins in our dataset; (2) measuring the variability in protein level and/or modifications between different cell types; (3) correlating disease state or phenotypes with protein levels and modifications; and (4) finding sources of translational regulation by comparing mRNA and protein levels. We have chosen a set of proteins that includes genes involved in and induced by wnt signaling, all kinases, all phosphatases, and genes involved in the cell cycle; we will measure the levels and modifications of these in hepatocarcinoma cell lines, colorectal cancer lines, and an immortalized retinal epithelium line. The data we deliver will be an important resource for many labs working on the cell biology of cancer. PUBLIC HEALTH RELEVANCE: We propose a project to accurately measure the levels of 1,000 proteins, and at the same time detect and quantitate all the modifications on these proteins in six different cell lines. These data should provide a very significant increase in our understanding of the behavior of these proteins, especially in cancer cells, open up a new type of approach to understanding how biological systems work, and provide a novel route to the creation of biomarkers for disease.