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. MS³ 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.

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我们的科学兴趣涉及通过专有质谱从头测序非胰岛天然两栖动物皮肤肽，包括具有分子内S–S键的自然肽。通过质谱分析在肽测序过程中可靠区分异构亮氨酸/异亮氨酸残基代表了这些化合物的一级结构完全自动化的工作流程中的瓶颈。MS ³能够解决此问题。较早前，我们证明了ETD-HCD方法区分单个肽中的Leu / Ile的先进效率，方法是连续将ETD应用于多质子化的肽，然后将HCD应用于手动选择的主要z-离子在其N末端具有目标异构体残基，并记录特征性w离子。后来，这种方法扩展到可以处理几个（4–7）宽带质量范围，而无需对原始z离子进行特殊隔离。本论文展示了将EThcD应用于整个质量范围内的天然非胰蛋白酶肽的复杂混合物而无需分离和中间分离目标z-肽的方法的高级版本离子。拟议的EThcD方法显示具有完整二硫键的大型天然肽的效率超过81％，同时抑制了自由基位点迁移的干扰过程。由于具有较高的速度和灵敏度，因此所提出的EThcD方法简化了分析过程，并实现了整个实验和数据处理的自动化。而且，在某些情况下，它提供了建立完整分子内二硫键环中残基性质的机会。

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