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.

自下而上蛋白质组学（bottom-up proteomics）方法已成为生物研究诸多领域的核心研究策略，酶解技术则是该技术体系的关键支撑环节。精氨酸内切酶（Endopeptidase Arg-C）可催化精氨酸残基羧基端的肽键水解，是自下而上蛋白质组学中一类重要的蛋白酶。然而，由于其酶解特异性不足且制备成本高昂，该酶的实际应用十分有限。本研究针对实际的Arg-C酶解场景，引入并优化了可逆氨基衍生化方法（柠康酰化（citraconylation）与去柠康酰化（decitraconylation）），并将其与胰蛋白酶（trypsin）解相结合。为优化Arg-C酶解，我们将该衍生化与胰蛋白酶解的联合策略命名为iArg-C酶解。相较于传统Arg-C酶解，iArg-C酶解可使肽段鉴定数量提升64.2%（11925 ± 199 vs 7262 ± 59），且酶切特异性显著提升（95.6% vs 73.6%）。将iArg-C酶解与目前广泛应用的胰蛋白酶、赖氨酸内切酶（Lys-C）酶解进行对比后发现，iArg-C的酶解效果略优于赖氨酸内切酶，但仍不及胰蛋白酶。综上，本研究建立的iArg-C酶解方法是一种极具应用前景的蛋白质组学研究策略，可作为胰蛋白酶解的优先替代方案，以实现更高的蛋白质组覆盖度。本研究相关数据已通过蛋白质组学数据共享平台（ProteomeXchange）公开，数据集编号为PXD007994。