C‑Terminal Charge-Reversal Derivatization and Parallel Use of Multiple Proteases Facilitates Identification of Protein C‑Termini by C‑Terminomics

The
identification of protein C-termini in complex proteomes is
challenging due to the poor ionization efficiency of the carboxyl
group. Amidating the negatively charged C-termini with ethanolamine
(EA) has been suggested to improve the detection of C-terminal peptides
and allows for a directed depletion of internal peptides after proteolysis
using carboxyl reactive polymers. In the present study, the derivatization
with N,N-dimethylethylenediamine (DMEDA) and (4-aminobutyl)­guanidine
(AG) leading to a positively charged C-terminus was investigated.
C-terminal charge-reversed peptides showed improved coverage of b-
and y-ion series in the MS/MS spectra compared to their noncharged
counterparts. DMEDA-derivatized peptides resulted in many peptides
with charge states of 3+, which benefited from ETD fragmentation.
This makes the charge-reversal strategy particularly useful for the
analysis of protein C-termini, which may also be post-translationally
modified. The labeling strategy and the indirect enrichment of C-termini
worked with similar efficiency for both DMEDA and EA, and their applicability
was demonstrated on an E. coli proteome.
Utilizing two proteases and different MS/MS activation mechanisms
allowed for the identification of >400 C-termini, encompassing
both
canonical and truncated C-termini.