Skin secretion of amphibians often represents the only weapon of these species against pathogens and predators. Peptides constitute the major portion of active molecules of that weapon and may be treated as potential pharmaceuticals for future generations. The first step of their efficient use involves establishing of their primary structure, i.e., sequencing. De novo sequencing by means of mass spectrometry was applied to Rana arvalis species, collected in the spring 2021 in Central Slovenia (vicinity of Ljubljana). HPLC-ESI-HRMS/MS with Orbitrap instruments was used to establish the skin peptidome of these species and compare it with the earlier identified skin peptidome of the Moscow population of Rana arvalis. Application of CID, HCD, ETD, and EThcD enabled detecting and sequencing 18 peptides; five of them were novel and may be treated as possible biomarkers of the Ljubljana population of Rana arvalis. Interestingly, representatives of two peptide families (temporins and brevinins 2) were not found in the Moscow population. MS³ modes, first of all EThcD, demonstrated their great potential in the de novo sequencing, including extraction of the sequence information from the intact peptides with disulfide cycle (rana box) in their structure and differentiation of isomeric Leu/Ile residues. Thus, all six isomeric residues were reliably distinguished in the novel melittin-related peptide AK-23-1. In addition, another post-translational modification dealing with carbonylation of the N-terminal Gly of novel temporin AVa was established using the MS³ mode. The obtained results demonstrate the efficiency of the use of MS³ tools in proteomics/peptidomics.

中文翻译：

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基于皮肤肽组组成的 Rana arvalis 种群的质谱区分

两栖动物的皮肤分泌物通常是这些物种对抗病原体和捕食者的唯一武器。肽构成了该武器活性分子的主要部分，可能被视为未来几代人的潜在药物。有效使用它们的第一步涉及建立它们的主要结构，即测序。2021 年春季在斯洛文尼亚中部（卢布尔雅那附近）采集的Rana arvalis物种应用了质谱从头测序。使用带有 Orbitrap 仪器的 HPLC-ESI-HRMS/MS 来建立这些物种的皮肤肽组，并将其与莫斯科林蛙种群早期鉴定的皮肤肽组进行比较. CID、HCD、ETD和ETHcD的应用实现了18种多肽的检测和测序；其中五个是新的，可能被视为卢布尔雅那Rana arvalis 种群的可能生物标志物。有趣的是，在莫斯科人群中没有发现两个肽家族（temporins 和 brevinins 2）的代表。MS ³模式，首先是 ETHcD，展示了它们从头开始的巨大潜力测序，包括从结构中具有二硫键循环（rana box）的完整肽中提取序列信息，以及区分异构 Leu/Ile 残基。因此，在新型蜂毒肽相关肽 AK-23-1 中，所有六个异构残基都得到了可靠区分。此外，使用 MS ³模式建立了另一个涉及新型 temporin AVa 的 N 端 Gly 羰基化的翻译后修饰。获得的结果证明了在蛋白质组学/肽组学中使用 MS ^{3工具的效率。}