Engineering Proteases for Mass Spectrometry‐Based Post Translational Modification Analyses

Abstract

Protein post‐translational modifications (PTMs) are important modulators of virtually all cellular processes, and frequently correlate with not only the rate but also severity of diseases. There has been considerable interest to map all possible PTM sites to be used as drug targets. Current approaches for PTM analysis suffer from a number of challenges; one of which is the lack of a PTM specific cleaving reagent. A central technology for global quantitative PTM analysis, mass spectrometry (MS) based proteomics, is biased toward trypsin due to its high activity and specificity. This bias becomes a problem when a PTM is located at or near tryptic cleavage sites, in which case the PTM might block recognition by trypsin, resulting in missed cleavage and sequence coverage gaps. Reviewed here are recent advances in engineering new proteases for PTM analyses, and how these new proteases are beginning to address current challenges in the field.

High-Recovery Desalting Tip Columns for a Wide Variety of Peptides in Mass Spectrometry-based Proteomics

“…Specifically, while 1158 unique peptides were identified using SDB-XC-tip and 4062 peptides were commonly identified, 6835 unique peptides were identified using ChocoTip (Figure 2c, Figure S2). The unique peptides identified in ChocoTip tended to be longer than the commonly identified peptides and unique peptides in SDB-XC-tip (Figure S3), suggesting that ChocoTip might be useful for bottom-up proteomics using long peptides, such as in post-translational modification analysis 47 and structural proteomics using limited proteolysis, 48 as well as middle-down proteomics. 49 Furthermore, the peptide intensity of each commonly identified peptide was consistently higher for ChocoTip over the entire retention time range (Figure 2d) and the trend toward greater intensity was more pronounced at longer retention times.…”

Proteomics and Precision Medicine

“…Based on the advantages of proteomics to determine post‐translational modification sites, phosphor‐proteomics is the most widely studied modified omics. With the endeavors over the decade, it is ordinary to identify over 10 000 phospho‐sites for a cell line and tissue.…”