High Throughput Quantitative Top-down Proteomics

In the post-genomic era, protein post-translational modifications (PTMs) have become crucial for regulating and diversifying the biological activity of proteins. Various PTMs on the same protein often interact with each other—a phenomenon known as PTM crosstalk—driving many signaling pathways. Therefore, analyzing intact proteoforms is essential for understanding the significance of different PTMs and their interactions. The main objective of my research program is to establish high-throughput quantitative top-down proteomics platforms that integrate top-down mass spectrometry (TD-MS) with innovative sample handling and separation technologies. We have developed methods for high-resolution reversed-phase liquid chromatography (RPLC) to analyze complex protein mixtures, such as cell lysates and serum samples, using both high pH and low pH separations. Additionally, we have optimized a quantitative top-down proteomics workflow utilizing intact protein-level tandem mass tag (TMT) labeling. To further enhance our capabilities, we created a novel analytical tool called the “spray-capillary,” which facilitates quantitative ultra-low volume sampling—down to picoliter (pL) volumes and sub-nanogram (ng) sample amounts. The spray-capillary enables single-cell metabolomics or proteomics by directly coupling capillary electrophoresis (CE) separation with mass spectrometry (MS) detection, without the need for additional devices. Overall, we believe these techniques represent innovative analytical tools that can be applied in the rapidly expanding field of real-life proteomics.