Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?
Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?
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化学介导的消化：中下蛋白质组学的替代领域？
DOI：
10.1021/acs.jproteome.7b00834
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发表时间：
2018
影响因子：
4.4
通讯作者：
Y. O. Tsybin
中科院分区：
文献类型：
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作者：
K. Srzentić;Konstantin O. Zhurov;A. Lobas;Gennady Nikitin;L. Fornelli;M. Gorshkov;Y. O. Tsybin
基于质谱仪(MS)的自下而上蛋白质组学中的蛋白质消化主要针对赖氨酸和精氨酸残基，主要产生0.6-3 kDa的多肽，用于所有主要王国生物的蛋白质组。MS技术的最新进展使得以高通量方式分析越来越长的(>；3 kDa)多肽的复杂混合物支持了中间向下蛋白质组学(MDP)方法的发展。产生更长的多肽是启动MDP管道的关键一步，但选择一种能够提供所需3-15 kDa多肽的裂解剂仍然是一个悬而未决的问题。最近的生物信息学研究表明，对甲硫氨酸(Met)、色氨酸(Trp)或半胱氨酸(Cys)等罕见氨基酸残基的切割适合于MDP方法。有趣的是，化学介导的蛋白水解酶唯一地允许靶向这些稀有氨基酸，对于这些稀有氨基酸来说，没有特定的蛋白水解酶是已知的。在这里，我们研究了以前报道的主要针对蛋氨酸、色氨酸和半胱氨酸残基的化学试剂的性能：氰溴、BNPS-skatole(3-bromo-3-methyl-2-(2-nitrophenyl)sulfanylindole)，和NTCB(2-硝基-5-硫代苯甲酸)。讨论了诸如消化重复性、多肽大小分布和副反应的发生等优点系数。基于NTCB的MDP工作流表现出了特别有吸引力的性能，NTCB被提出作为一种潜在的MDP开发裂解剂。
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.