Quantitative Top-down Proteomics Revealed Kinase Inhibitor-Induced Proteoform-Level Changes in Cancer Cells.

Abstract

Quantitative analysis of proteins and their post-translational modifications (PTMs) in complex biological samples is critical to understanding cellular biology as well as disease detection and treatment. Top-down proteomics methods provide a "bird's eye" view of the proteome by directly detecting and quantifying intact proteoforms. Here, we developed a high-throughput quantitative top-down proteomics platform to probe intact proteoform and phosphoproteoform abundance changes in HeLa cells as a result of treatment with staurosporine (STS), a broad-spectrum kinase inhibitor. In total, we identified and quantified 1187 proteoforms from 215 proteoform families. Among them, 55 proteoforms from 37 proteoform families were significantly changed upon STS treatment. These proteoforms were primarily related to catabolic, metabolic, and apoptotic pathways that are expected to be impacted as a result of kinase inhibition. In addition, we manually evaluated 25 proteoform families that expressed one or more phosphorylated proteoforms. We observed that phosphorylated proteoforms in the same proteoform family, such as eukaryotic initiation factor 4E binding protein 1 (4EBP1), were differentially regulated relative to the unphosphorylated proteoforms. Combining relative profiling of proteoforms within these proteoform families with individual proteoform profiling results in a more comprehensive picture of STS treatment-induced proteoform abundance changes that cannot be achieved using bottom-up methods.