Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?

Protein digestion in mass spectrometry (MS)-based bottom-up proteomics targets mainly lysine and arginine residues, yielding primarily 0.6–3 kDa peptides for the proteomes of organisms of all major kingdoms. Recent advances in MS technology enable analysis of complex mixtures of increasingly longer (>3 kDa) peptides in a high-throughput manner supporting the development of a middle-down proteomics (MDP) approach. Generating longer peptides is a paramount step in launching an MDP pipeline, but the quest for the selection of a cleaving agent that would provide the desired 3–15 kDa peptides remains open. Recent bioinformatics studies have shown that cleavage at the rarely occurring amino acid residues such as methionine (Met), tryptophan (Trp), or cysteine (Cys) would be suitable for MDP approach. Interestingly, chemical-mediated proteolytic cleavages uniquely allow targeting these rare amino acids, for which no specific proteolytic enzymes are known. Herein, as potential candidates for MDP-grade proteolysis, we have investigated the performance of chemical agents previously reported to target primarily Met, Trp, and Cys residues: CNBr, BNPS-Skatole (3-bromo-3-methyl-2-(2-nitrophenyl)­sulfanylindole), and NTCB (2-nitro-5-thiobenzoic acid), respectively. Figures of merit such as digestion reproducibility, peptide size distribution, and occurrence of side reactions are discussed. The NTCB-based MDP workflow has demonstrated particularly attractive performance, and NTCB is put forward here as a potential cleaving agent for further MDP development.

基于质谱（MS）的鸟枪法蛋白质组学（bottom-up proteomics）的蛋白质消化主要靶向赖氨酸与精氨酸残基，对于所有主要生物界的生物体蛋白质组而言，其消化产物主要为0.6~3 kDa的肽段。近年来质谱技术的进步使得能够以高通量方式分析长度更长（>3 kDa）的复杂肽段混合物，这推动了中段肽段蛋白质组学（middle-down proteomics, MDP）方法的发展。生成更长肽段是构建MDP流程的关键步骤，但目前仍未找到可获得目标3~15 kDa肽段的理想切割试剂。近期的生物信息学研究表明，在罕见氨基酸残基如甲硫氨酸（Met）、色氨酸（Trp）或半胱氨酸（Cys）位点进行切割，适用于MDP方法。有趣的是，化学介导的蛋白水解切割能够特异性靶向这些罕见氨基酸——目前尚未发现针对这些位点的特异性蛋白水解酶。本文中，我们以可用于MDP级蛋白水解的潜在试剂为研究对象，评估了此前报道的分别靶向甲硫氨酸、色氨酸与半胱氨酸残基的三种化学试剂的性能：溴化氰（CNBr）、3-溴-3-甲基-2-(2-硝基苯基)硫代吲哚（BNPS-Skatole）以及2-硝基-5-硫代苯甲酸（NTCB）。本文还讨论了消化重现性、肽段尺寸分布以及副反应发生情况等性能指标。基于NTCB的MDP工作流程展现出尤为优异的性能，因此本文提出将NTCB作为可用于后续MDP开发的潜在切割试剂。