Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russia.
Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia.
J Am Soc Mass Spectrom. 2018 May;29(5):842-852. doi: 10.1007/s13361-017-1857-y. Epub 2018 Jan 3.
Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S-S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4-7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops. Graphical Abstract ᅟ.
我们的科学兴趣涉及从头测序非胰蛋白酶天然两栖动物皮肤肽，包括那些具有分子内 S-S 键的肽，方法是仅使用质谱法。在肽测序过程中，通过质谱可靠地区分异构亮氨酸/异亮氨酸残基是这些化合物一级结构阐明完全自动化工作流程中的一个瓶颈。MS 能够解决这个问题。我们之前证明了 ETD-HCD 方法在连续应用 ETD 对多质子化肽，然后对具有靶向 N 末端异构残基的手动选择的初级 z-离子应用 HCD，并记录特征 w-离子的情况下，对单个肽中 Leu/Ile 进行区分的高级效率。后来，这种方法被扩展到处理几个（4-7）宽波段质量范围，而无需对初级 z-离子进行特殊分离。本文演示了在整个质量范围内将 EThcD 应用于复杂的天然非胰蛋白酶肽混合物而不进行分离和中间分离靶向 z-离子的情况下的这种方法的高级版本。所提出的 EThcD 方法对具有完整二硫键的大型天然肽显示出超过 81%的效率，同时抑制了自由基位点迁移的干扰过程。由于速度更快、灵敏度更高，所提出的 EThcD 方法简化了分析过程，并允许整个实验和数据处理的自动化。此外，在某些情况下，它还有机会确定完整分子内二硫键环中残基的性质。
