Developing Proteomic Methods to Assay Function of Proteoforms

Abstract

The human genome encodes a repertoire of ~20,000 proteins. The unique functional roles of these proteins diversify through mechanisms of allelic variation, post-translational modifications (PTMs), alternative splicing, and protein cleavages. The modified versions of a protein, or proteoforms, comprise a vast collection of molecules originating from a single protein-coding gene. Proteoforms can perform different functions and warrant extensive characterization for their involvement in normal cell function and disease. Modern advances in genomic sequencing and mass spectrometry (MS) have accelerated our capacity to identify proteoforms at scale. However, a vast majority of proteoforms lack functional annotation. In this thesis work, I developed novel MS-based proteomic methods to assess the impact of PTMs, missense mutations, and protein cleavages on protein function at scale. I chose to measure thermal stability and turnover of proteins to probe functional differences caused by protein modifications. Measuring these protein properties is feasible at the proteome scale and they encapsulate many generalized functions of PTMs, mutations, and protein cleavages. To capture proteoform functions, I performed our molecular selections at the protein-level, thus retaining proteoform-specific turnover and stability information and MS readout at the peptide-level. I identified functional proteoforms by comparing proteoform-specific peptides to their cognate peptides from their unmodified proteoform. For the PTM phosphorylation, we identified 253 and 68 phosphorylated proteoforms that alter protein turnover or thermal stability in the yeast proteome, respectively. For protein cleavages, we implemented protein turnover and thermal stability assays to identify and functionally characterize substrates of the NSP5 protease from SARS-CoV-2. For protein mutations, we piloted a peptide barcoding method to represent each protein variant with a short peptide and to enable pooled functional screens for protein variant libraries by MS. Lastly, my thesis work expanded to implementing novel MS acquisition strategies and developing software to improve peptide detection and quantitative precision. Collectively, these high-throughput proteomic methods enabled biochemical interrogation of proteoforms, accelerating the functional characterization of the modified proteome.