Exopeptidase assisted N- and C-terminal proteome sequencing

ABSTRACT: Due to mechanisms such as alternative translation starts or proteolytic processing, in-vivo proteoforms do not necessarily correspond to those encoded in the genome. Therefore, knowledge of protein termini is an indispensable prerequisite to understand protein function. Sequencing of protein N- and C-termini has been limited to single purified protein species, while the proteome-wide identification of N- and C-termini relies on the generation of single, terminal proteotypic peptides followed by chemical enrichment or depletion strategies to facilitate their detection via mass spectrometry (MS). To overcome limitations in such approaches, we present a novel concept that readily enables unbiased ladder sequencing of protein N- and C-termini. The approach is based on the combination of exopeptidase digestions and two-dimensional separation by strong cation exchange chromatography and reversed-phase liquid chromatography coupled online to tandem-MS. We demonstrate the potential of this methodology by analyzing the N- and C-terminome of S. cerevisiae, identifying 2,189 N-termini, and 1,562 C-termini. In conclusion, the presented method largely expands the proteomics toolbox enabling N- and C-terminal sequential characterization of entire proteomes.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Andreas Tholey  

LAB HEAD: Prof. Dr. Andreas Tholey

PROVIDER: PXD015612 | Pride | 2025-10-15

REPOSITORIES: Pride