Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory 1 Bld. 3, 119991, Moscow, Russia.
J Am Soc Mass Spectrom. 2017 Aug;28(8):1600-1611. doi: 10.1007/s13361-017-1674-3. Epub 2017 Apr 26.
An EThcD-based approach for the reliable discrimination of isomeric leucine and isoleucine residues in peptide de novo sequencing procedure has been proposed. A multistage fragmentation of peptide ions was performed with Orbitrap Elite mass spectrometer in electrospray ionization mode. At the first stage, z-ions were produced by ETD or ETcaD fragmentation of doubly or triply charged peptide precursor ions. These primary ions were further fragmented by HCD with broad-band ion isolation, and the resulting w-ions showed different mass for leucine and isoleucine residues. The procedure did not require manual isolation of specific z-ions prior to HCD stage. Forty-three tryptic peptides (3 to 27 residues) obtained by trypsinolysis of human serum albumin (HSA) and gp188 protein were analyzed. To demonstrate a proper solution for radical site migration problem, three non-tryptic peptides were also analyzed. A total of 93 leucine and isoleucine residues were considered and 83 of them were correctly identified. The developed approach can be a reasonable substitution for additional Edman degradation procedure, which is still used in peptide sequencing for leucine and isoleucine discrimination. Graphical Abstract ᅟ.
提出了一种基于 EThcD 的方法，用于在从头测序肽的过程中可靠地区分异构的亮氨酸和异亮氨酸残基。采用 Orbitrap Elite 质谱仪在电喷雾电离模式下对肽离子进行多级碎裂。在第一阶段，通过 ETD 或 ETcaD 碎裂双电荷或三电荷肽前体离子产生 z 离子。这些初级离子进一步通过 HCD 进行宽频带离子隔离碎裂，得到的 w 离子显示亮氨酸和异亮氨酸残基的不同质量。该方法在进行 HCD 阶段之前不需要手动分离特定的 z 离子。分析了人血清白蛋白 (HSA) 和 gp188 蛋白胰蛋白酶水解得到的 43 个肽（3 至 27 个残基）。为了证明自由基位点迁移问题的合理解决方案，还分析了三个非胰蛋白酶肽。共考虑了 93 个亮氨酸和异亮氨酸残基，其中 83 个被正确鉴定。所开发的方法可以替代额外的 Edman 降解程序，该程序仍用于亮氨酸和异亮氨酸的肽测序区分。
