Advancing top-down proteomics with capillary electrophoresis-mass spectrometry

Project Summary The research of the Sun group can be divided into two directions. In the first direction, the Sun group focuses on the development of novel capillary electrophoresis-mass spectrometry (CE-MS) techniques to advance bottom-up proteomics (BUP, peptide-centric), top-down proteomics (TDP, proteoform-centric), and native proteomics (protein complex-centric) towards complete proteome coverage of human cells with single-cell resolution. CE-MS is a powerful analytical tool for the highly sensitive characterization of peptides, proteoforms, and protein complexes in complex biological samples. In the second direction, the Sun group is applying the CE-MS techniques to developmental biology and cancer biology. For developmental biology, we are pursuing a better understanding of the roles played by specific proteins, proteoforms, and protein complexes in modulating the important events during early vertebrate embryogenesis (i.e., early cellular differentiation and zygotic genome activation (ZGA)). For cancer biology, we aim to discover new proteoform biomarkers of colorectal cancer (CRC) metastasis by quantitative TDP studies of CRC cell lines and tumors. In this MIRA application, the Sun group will advance CE-MS-based TDP techniques to achieve the first draft of the proteoform atlas of a human cancer cell line (i.e., HeLa cells) and enable TDP measurement of a few even single human cells. The Sun group will also apply the novel techniques to developmental biology to discover crucial proteoforms that modulate early cellular differentiation and ZGA during early vertebrate embryogenesis via quantitative TDP studies of zebrafish embryos and blastomeres. The proteoform atlas of HeLa cells will contain at least one intact proteoform per protein-coding gene, and it will be an extremely valuable resource for fundamental and translational research. The generation of the first proteoform atlas of human cells will be a pivotal step toward the “Human Proteoform Project”. The novel CE-MS techniques will play crucial roles in advancing TDP toward the comprehensive characterization of proteoforms in various biological systems. The TDP studies of zebrafish embryos and blastomeres will provide new insights into the roles played by specific proteoforms in modulating early cellular differentiation and ZGA. The single-cell TDP technique can be applied to various biological fields, e.g., cancer biology, developmental biology, and neuroscience, to better our understanding of cell-to-cell heterogeneity.