CAREER: Nanoflow RPLC-CZE-MS/MS: a novel approach for deep and highly sensitive bottom-up proteomics

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In this project, researchers at the Michigan State University will develop novel methods that transform a mass spectrometry-based analytical platform so that it can identify and quantify protein molecules from biological samples where they currently cannot be detected. The novel platform will provide much greater sensitivity and specificity, in part by developing higher separation capacities than the commonly used mass spectrometry-based methodologies. The impact of these advances will be wide, as many areas of fundamental research in biology involve mass-limited protein analyses, such as developmental biology, cancer biology, and neuroscience. The project requires advances in both chemistry and biology, and through involvement in the research it will train a new generation of graduate and undergraduate students for careers in interdisciplinary research. Project personnel will host local high school students from under-represented groups as participants in the project, nurturing their interest in science, prompting active learning experiences, and encouraging them to pursue science careers.Bottom-up proteomics is a powerful tool for large-scale and high-throughput characterization of proteins in the cell. Over 8000 protein identifications from human cells can be accomplished using advanced bottom-up proteomic methodologies, given hundreds of micrograms of protein material. However, to progress in using bottom-up proteomics for the analysis of mass-limited proteome samples, containing only low micrograms to nanograms of proteins, will require novel analytical tools with drastically better sensitivity. This project will develop an innovative and automated nanoflow reversed-phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry (nanoRPLC-CZE-MS/MS) platform for highly sensitive and deep bottom-up proteomics. This transformative approach will boost the sensitivity of bottom-up proteomics by over two orders of magnitude, enabling the identification of about 8000 proteins from only nanograms of human cell protein material as proof of concept. The novel methodology will benefit a variety of scientific communities for pursuing answers to important fundamental questions that involve mass-limited proteome analyses. The research will also apply the nanoRPLC-CZE-MS/MS technology in discovering the changes in the level of abundance of chromatin-associated proteins in zebrafish embryos during the maternal-to-zygotic transition (MZT). The MZT is key for development, and it coordinates important events during early embryogenesis, e.g., zygotic genome activation. The quantitative proteomic dataset will provide a better understanding of the roles played by chromatin-associated proteins in modulating the zygotic genome activation during the MZT. Results of the project can be found at https://sungroupchemmsu.wixsite.com/sungroup/publications.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.