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.