P12: Improved C-terminomics for protease substrate discovery

Imporved C-terminomics for protease substrate discovery (PI Pitter Huesgen)

Limited proteolytic processing changes activity, location, interactions and function of substrate protein by site-specific cleavage. The resulting shorter proteoforms are delimited by distinctive protease-generated neo-N- and neo-C-termini. We and others (1) have developed efficient protocols that enable identification of proteolytic processes and protease substrates based on N-termini enrichment, even in limited samples (2). However, protein N-termini do not reveal information on carboxypeptidase activities or processing sites in type I membrane proteins fragments released by ectodomain shedding, an important cellular signaling process (1). In addition, many relevant cleavage events will not be detected if N-terminal peptides are too short, too long or contain amino acid compositions that impair ionization. Methods for complementary profiling of protein C-termini are therefore highly desired.

With Oliver Schilling (P10), we have developed the first strategy for unbiased enrichment of C-termini by negative selection more than a decade ago (3, see Figure). However, this workflow was time-consuming and loss-intensive an was rarely applied. Here we aim to develop promising approaches described in the recent literature into a robust, ideally automatable C-terminome enrichment strategy for application to caspase substrate discovery (with P4/ Häcker) and the identification of carboxypeptidase substrates (with P9/Reinheckel)

1.         Niedermaier S, Huesgen PF. Positional proteomics for identification of secreted proteoforms released by site-specific processing of membrane proteins. Biochimica et Biophysica Acta - Proteins and Proteomics. 2019;1867(12):140138.

2.         Weng SSH, Demir F, Ergin EK, et al. Sensitive Determination of Proteolytic Proteoforms in Limited Microscale Proteome Samples. Molecular & Cellular Proteomics. 2019 Nov;18(11):2335-2347.

3.         Schilling O, Barre O, Huesgen PF, et al. Proteome-wide analysis of protein carboxy termini: C terminomics. Nature methods. 2010 Jul;7(7):508-11.

Figure caption: CTAILS, the first method for unbiased enrichment and analysis of C-terminal peptides. Modified from (3).