Combination of SCX Fractionation and Charge-Reversal Derivatization Facilitates the Identification of Nontryptic Peptides in C‑Terminomics

The
proteome wide, mass spectrometry based identification of protein
C-termini is hampered by factors such as poor ionization efficiencies,
low yielding labeling strategies, or the need for enrichment procedures.
We present a bottom-up proteomics workflow to identify protein C-termini
utilizing a combination of strong cation exchange chromatography,
on-solid phase charge-reversal derivatization and LC–MS/MS
analysis. Charge-reversal improved both MS and MS/MS spectra quality
of peptides carrying nonbasic C-terminal residues, allowing the identification
of a high number of noncanonical C-termini not identified in nonderivatized
samples. Further, we could show that C-terminal 18O labeling
introduced during proteolytic processing of the samples is not suitable
to distinguish internal from C-terminal peptides. The presented workflow
enables the simultaneous identification of proteins by internal peptides
and additionally provides data for the C- and N-terminome. Applying
the developed workflow for the analysis of a Saccharomyces
cerevisiae proteome allowed the identification of 734 protein
C-termini in three independent biological replicates, and additional
789 candidate C-termini identified in two or one of three biological
replicates, respectively. The developed analytical workflow allowed
us to chart the nature of the yeast C-terminome in unprecedented depth
and provides an alternative methodology to assess C-terminal proteolytic
protein processing.