Post-Translational Modifications Enrichment Analysis
Post-translational modifications (PTMs) are critical for normal protein function, perturbed in disease, and can elicit changes in genome function (epigenetics) that are not mediated through a change in the DNA sequence itself. Genomics has provided a
vast amount of information linking gene activity with disease, but it does not predict what PTMs proteins may undergo; LC-MS is both highly sensitive and specific, allowing numerous types of PTMs, such as phosphorylation, ubiquitination, acetylation,
and glycosylation, to be studied in an “omic” manner (Figure 1A). However, their detection is challenging since PTMs occur sub-stoichiometrically. This challenge is overcome using enrichment strategies specific for the
PTM of interest. For example, ubiquitin is a highly conserved regulatory protein whose C-terminus can be covalently attached to target proteins and plays a critical role in biological processes regulating protein degradation and function. Dysregulation
of ubiquitination is associated with diseases, including cancer, immune disorders, and neurological disease. Proteolysis using trypsin to cleaves after arginine and lysine residues, which conveniently cleaves ubiquitin and leaves behind a di-glycine
tag (K-ɛ-GG) (Figure 1B). Leveraging commercially available antibodies (CST) recognizing K-ɛ-GG can enrich these peptides and permit analysis by LC-MS/MS. We show you need relatively high starting amounts of protein (10mg). However,
this also allows for enrichment of other PTMs using the same starting material (Pandey et al, CHM, 2017, Tidmore et al, IJMS, 2021). Here we show phosphopeptide enrichment from 100 μg of peptides from the flowthrough of the Ubi enrichment.