High-throughput Quantitative Top-down Proteomics

Protein post-translational modifications (PTMs) have emerged in the post-genomic era as critical features in regulating and diversifying protein biological activity. In addition, various PTMs on the same protein tend to physically interact with each other, PTM crosstalk, that drives many signaling pathways. Thus, analysis of intact proteoforms is necessary for in-depth knowledge of the significance of various PTMs and related crosstalk. We have developed methods for high-resolution reversed-phase liquid chromatography (RPLC)-based separations (high pH and/or low pH) for analysis of complex protein mixtures such as cell lysates and serum samples. We also established and optimized a quantitative top-down proteomics workflow using intact protein-level tandem mass tag (TMT) labeling. We have applied our LC-MS-based quantitative top-down proteomics for several biological applications, including (1) Analyzing serum autoantibodies in patients with systemic lupus erythematosus (SLE), which holds the potential to provide new insights regarding the autoantibody affinity maturation process and may lead to novel biomarkers for the diagnosis and treatment of SLE; (2) Probing intact proteoform and phosphoproteoform abundance changes in HeLa cells as a result of treatment with staurosporine (STS), a broad-spectrum kinase inhibitor.