Developing new technology and novel strategies to enhance the quality of quantitative proteomic analyses

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For over two decades, mass spectrometry (MS) has proven to be particularly well suited to analyze proteins. Initial efforts in this field successfully identified dozens of proteins in a single experiment and it is now possible to identify thousands of proteins from complicated biological extracts with relative ease. MS-based protein characterization (proteomics) studies have become increasingly prevalent and attention has begun to shift from merely identifying the presence of proteins in a given sample to characterizing the dynamics of proteins in the context of a changing biological system. Although a number of strategies to quantify protein dynamics using MS are available, many are cost prohibitive or negatively affect the analytical sensitivity. Our ongoing research program will strive to develop technology and strategies to improve the reproducibility and overall quality of quantitative proteomic analyses; this work will concurrently develop improved protein identification methods as well. To begin, we will investigate the dynamics of protein phosphorylation, an important protein modification that is employed by cells to control protein function. To date, relatively few predicted phosphorylation sites have been experimentally confirmed despite the fact that many different disease states are associated with faulty phosphorylation mechanics. Current methods to chemically modify and enrich phosphorylated peptides involve multiple transfer steps to containers with relatively large volumes, resulting in sample loss and depletion of analytical sensitivity. To overcome this, microliter-scale columns packed with peptide-binding resin will be used to concentrate and process complicated biological samples so that phosphorylated peptides may be isolated with minimal transfer steps and user intervention. Furthermore, the ability to chemically modify peptides in distinct ways will be available which will allow peptide/phosphopeptide concentration dynamics to be measured over the course of an external stimulus (e.g. determining the proteomic response of cells exposed to a drug). The application of this technology to probe specific biological questions will be carried out in parallel with its development.