Carboxypeptidase B‑Assisted Charge-Based Fractional Diagonal Chromatography for Deep Screening of C‑Terminome

The determination
of protein C-termini is of great significance
for protein function annotation and proteolysis research. However,
the progress of C-terminomics is still far behind its counterpart,
N-terminomics, because of the low reactivity of the carboxyl group.
Herein, we developed a negative selection strategy, termed carboxypeptidase
B-assisted charge-based fractional diagonal chromatography (CPB-ChaFRADIC),
to achieve a global C-terminome analysis. The highly reactive carboxypeptidase
B cleavage was utilized to reduce the charge state of non-C-terminal
peptides. Together with high-performance charge-based fractional diagonal
chromatography, the C-terminal peptides could be isolated. Such a
strategy was applied for profiling C-termini from Escherichia
coli cell lysates and 441 canonical C-termini and 510 neo-C-termini
originating from proteolytic processing were identified. These findings
represent 2-fold and 5.8-fold that of identified C-termini via direct
analysis, respectively. Using parallel digestion with trypsin and
LysC, such a strategy enabled the identification of 604 canonical
C-termini and 818 neo-C-termini, representing the largest C-terminome
data set of E. coli, and no deficiency in His/Lys/Arg-containing
C-terminal peptides was observed. The presented CPB-ChaFRADIC strategy
is therefore a highly efficient and unbiased strategy for large-scale
C-terminome analysis. Furthermore, using the CPB-ChaFRADIC strategy,
we identified 107 cleavage sites and 102 substrates of caspase-3 in
Jurkat cells, demonstrating that the CPB-ChaFRADIC strategy shows
great promise in promoting proteolysis research. Data are available
via ProteomeXchange with identifier PXD018520.