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Combination of SCX fractionation and charge-reversal derivatization facilitates the identification of non-tryptic 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 non-basic C-terminal residues, allowing the identification of a high number of non-canonical C-termini not identified in non-derivatized 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 S. 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.