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

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.