posted on 2020-03-24, 15:33authored byAndreas
O. Helbig, Andreas Tholey
Due to mechanisms such as proteolytic processing or alternative
translation starts, in vivo proteoforms do not necessarily correspond
directly to those encoded in the genome. Therefore, the knowledge
of protein termini is an indispensable prerequisite to understand
protein functions. So far, 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 the numerous limitations in such approaches, we present
an alternative concept that readily enables unbiased ladder sequencing
of protein N- and C-termini. The approach combines exopeptidase digestions
of the proteome with two-dimensional chromatographic separation and
tandem-MS. We demonstrate the potential of the methodology by analyzing
the N- and C-terminome of S. cerevisiae, identifying
2190 N-termini and 1562 C-termini. In conclusion, the presented method
largely expands the proteomics toolbox enabling N- and C-terminal
sequential characterization of entire proteomes.