The critical protein actors in biological systems are the intact proteoforms, namely the different forms of proteins produced from the genome in a variety of splice forms and adorned with a myriad of post-translational modifications that dramatically affect their function. Lamentably, today's bottom-up proteomic technologies, which identify and quantify peptides derived from proteins, rather than the proteins themselves, fail to deliver crucial protein-level information to biologists. We address this problem here, by developing a new paradigm for proteomic analyses, which uses intact mass and lysine count to identify and quantify the proteoforms present in a biological sample. Our strategy integrates state-of-the-art proteomic and genomic technologies. We couple a new generation of high resolution mass spectrometers and a novel isotopic tagging strategy to yield for each proteoform its accurate mass, its number of lysines, and its relative abundance. Parallel RNA-Seq analyses on the same sample will provide transcriptomic data to construct a tightly focused sample-specific proteoform database. Comparison of the mass spectrometric data with the proteoform database will reveal the proteoform identities present in the sample, and the isotopic tagging strategy will provide relative abundance. This information will comprise a revolutionary new tool for biology.
The goal of this proposal is to provide scientists with new tools to identify and quantify the protein variations, at the whole protein level, expressed in eac individual sample under study. This information will facilitate improved understanding of the functional relevance of genetic variations and lead to advances in the diagnosis and treatment of complex diseases.
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