Natural product extracts present inherently complex matrices in which the identification of novel bioactive peptide species is challenged by low-abundance masses and significant structural and sequence diversity. Additionally, discovery efforts often result in the re-identification of known compounds, where modifications derived in vivo or during sample handling may obscure true sequence identity. Herein, we identify mass spectral (MS2) "fingerprint" ions characteristic of cyclotides, a diverse and biologically active family of botanical cysteine-rich peptides, based on regions of high sequence homology. We couple mass shift analysis with MS2 spectral fingerprint ions cross referenced with CyBase-a cyclotide database-to discern unique mass species in Viola communis extracts from mass species that are likely already characterized and those with common modifications. The approach is extended to a related class of cysteine-rich peptides, the trypsin inhibitors, using the characterized botanical species Lagenaria siceraria. Coupling the observation of highly abundant MS2 ions with mass shift analysis, we identify a new set of small, highly disulfide-bound cysteine-rich L. siceraria peptides.

中文翻译：

---

通过 MS/MS 指纹离子探索富含半胱氨酸的天然产物肽的多样性。

天然产物提取物存在固有的复杂基质，其中新的生物活性肽物种的鉴定受到低丰度质量和显着的结构和序列多样性的挑战。此外，发现努力通常会导致对已知化合物的重新识别，其中在体内或样品处理过程中产生的修饰可能会掩盖真实的序列同一性。在这里，我们基于高序列同源性区域鉴定了 cyclotides 的质谱 (MS2) “指纹”离子特征，这是一个多样化且具有生物活性的植物性富含半胱氨酸肽家族。我们将质量转移分析与 MS2 光谱指纹离子与 CyBase（一种环肽数据库）交叉引用相结合，以从可能已经表征的质量物种和具有常见修饰的质量物种中辨别 Viola Communis 提取物中的独特质量物种。该方法被扩展到一类相关的富含半胱氨酸的肽，即胰蛋白酶抑制剂，使用特征植物物种 Lagenaria siceraria。将高丰度 MS2 离子的观察与质量转移分析相结合，我们确定了一组新的小型、高度二硫键结合的富含半胱氨酸的 L. seceraria 肽。