Beyond Trypsin: Strategies to Improve Mass Spec Sequence Coverage and Post-Translation...

Beyond Trypsin: Strategies to Improve Mass Spec Sequence Coverage and Post-Translation Modification Analysis

ABSTRACT

Proteomics research largely focuses on using biological mass spectrometry (MS) to sequence individual proteins and whole proteomes. The target proteins are first digested with proteases, in which trypsin has historically been used due to its high activity, specificity, optimal size, and C-terminally charged peptides for MS analysis. While trypsin is highly effective for preparing samples which are easily “sequenced” using mass spectrometry, there are often portions of the protein that are not sequenced (covered) in the process. Importantly, the requirement for high protein sequence coverage is increasing, particularly for complex mixtures like serum. This demand is due, at least in part, to the stringency needed to identify disease markers, understand the role of protein modifications in disease, and implement personalized medicine. As such, additional proteases like Asp-N, chymotrypsin, Glu-c, Lys-C and others are utilized producing more complete protein sequence coverage. Furthermore, the identification of post-translation modifications (PTM) like phosphorylation, glycosylation, and lysine-acetylation, often requires other proteases because tryptic peptides fail to produce usable PTM peptides because of sequence constraints (i.e. peptide is too short or too long).

This webinar will review the common MS workflows which use proteases as well as other enzymes like glycosidases in proteomic analysis. We will present specific examples of using alternative proteases to increase sequence coverage and characterize PTM’s. These studies will highlight the requirement of proteolytic enzymes to characterize protein structure and study a wide range of biological problems including signal transduction, epigenetics, and membrane protein structure.