Quantitative\nTop-down\nProteomics Revealed Kinase Inhibitor-Induced\nProteoform-Level Changes in Cancer Cells

Quantitative analysis of proteins\nand their post-translational\nmodifications (PTMs) in complex biological samples is critical to\nunderstanding cellular biology as well as disease detection and treatment.\nTop-down proteomics methods provide a “bird’s eye”\nview of the proteome by directly detecting and quantifying intact\nproteoforms. Here, we developed a high-throughput quantitative top-down\nproteomics platform to probe intact proteoform and phosphoproteoform\nabundance changes in <i>HeLa</i> cells as a result of treatment\nwith staurosporine (STS), a broad-spectrum kinase inhibitor. In total,\nwe identified and quantified 1187 proteoforms from 215 proteoform\nfamilies. Among them, 55 proteoforms from 37 proteoform families were\nsignificantly changed upon STS treatment. These proteoforms were primarily\nrelated to catabolic, metabolic, and apoptotic pathways that are expected\nto be impacted as a result of kinase inhibition. In addition, we manually\nevaluated 25 proteoform families that expressed one or more phosphorylated\nproteoforms. We observed that phosphorylated proteoforms in the same\nproteoform family, such as eukaryotic initiation factor 4E binding\nprotein 1 (4EBP1), were differentially regulated relative to the unphosphorylated\nproteoforms. Combining relative profiling of proteoforms within these\nproteoform families with individual proteoform profiling results in\na more comprehensive picture of STS treatment-induced proteoform abundance\nchanges that cannot be achieved using bottom-up methods.