Reversible Lysine Derivatization Enabling Improved Arg‑C Digestion, a Highly Specific Arg‑C Digestion Using Trypsin

The bottom-up proteomics
approach has become an important strategy
in diverse areas of biological research, and the enzymatic digestion
is essential for this technology. Endopeptidase Arg-C catalyzing the
hydrolytic cleavage of peptide bonds C-terminal to arginine could
be an important protease in bottom-up proteomics. However, it has
been seldom applied due to its low specificity and high cost. In this
report, the reversible amine derivatization method (citraconylation
and decitraconylation) was introduced and optimized toward a real
Arg-C digestion using trypsin. Combination of the reversible derivatization
and trypsin digestion (termed iArg-C digestion for improved Arg-C
digestion) resulted in 64.2% more peptide identification (11 925
± 199 vs 7262 ± 59) and significantly higher cleavage specificity
(95.6% vs 73.6%) than the conventional Arg-C digestion. Comparison
of iArg-C digestion with the widely used trypsin and Lys-C digestion
revealed that iArg-C performed slightly better than Lys-C although
not comparable to trypsin. Therefore, the well-established iArg-C
digestion method is a promising approach for proteomics studies and
could be used as the prior alternative digestion method to trypsin
digestion in order to achieve higher proteome coverage. Data are available
via ProteomeXchange with identifier PXD007994.