Compared to their linear counterparts, cyclic peptides show better biological activities, such as antibacterial, immunosuppressive, and anti-tumor activities, and pharmaceutical properties due to their conformational rigidity. However, cyclic peptides could form numerous putative metabolites from potential hydrolytic cleavages and their fragments are very difficult to interpret. These characteristics pose a great challenge when analyzing metabolites of cyclic peptides by mass spectrometry. This study was to assess and apply a software-aided analytical workflow for the detection and structural characterization of cyclic peptide metabolites. Insulin and atrial natriuretic peptide (ANP) as model cyclic peptides were incubated with trypsin/chymotrypsin and/or rat liver S9, followed by data acquisition using TripleTOF® 5600. Resultant full-scan MS and MS/MS datasets were automatically processed through a combination of targeted and untargeted peak finding strategies. MS/MS spectra of predicted metabolites were interrogated against putative metabolite sequences, in light of a, b, y and internal fragment series. The resulting fragment assignments led to the confirmation and ranking of the metabolite sequences and identification of metabolic modification. As a result, 29 metabolites with linear or cyclic structures were detected in the insulin incubation with the hydrolytic enzymes. Sequences of twenty insulin metabolites were further determined, which were consistent with the hydrolytic sites of these enzymes. In the same manner, multiple metabolites of insulin and ANP formed in rat liver S9 incubation were detected and structurally characterized, some of which have not been previously reported. The results demonstrated the utility of software-aided data processing tool in detection and identification of cyclic peptide metabolites.

与线性对应物相比，环状肽由于其构象刚性而具有更好的生物学活性，例如抗菌，免疫抑制和抗肿瘤活性，以及​​药物特性。然而，环肽可通过潜在的水解裂解形成许多假定的代谢产物，并且其片段很难解释。当通过质谱分析环肽的代谢物时，这些特征提出了巨大的挑战。这项研究旨在评估和应用软件辅助的分析工作流程来检测和鉴定环肽代谢产物的结构。将胰岛素和心房利钠肽（ANP）作为模型环肽与胰蛋白酶/胰凝乳蛋白酶和/或大鼠肝S9一起孵育，然后使用5600进行数据采集。通过结合有针对性和无目标的峰发现策略，可以自动处理最终的全扫描MS和MS / MS数据集。根据a，b，y和内部片段序列，对假定的代谢物序列询问了预测的代谢物的MS / MS光谱。产生的片段分配导致代谢物序列的确认和排序以及代谢修饰的鉴定。结果，在胰岛素与水解酶的温育中检测到29种具有线性或环状结构的代谢物。进一步确定了二十种胰岛素代谢物的序列，这与这些酶的水解位点一致。以相同的方式，检测并在大鼠肝脏S9培养中形成了多种胰岛素和ANP代谢物，并对其结构进行了表征，其中一些以前没有被报道过。结果证明了软件辅助数据处理工具在检测和鉴定环肽代谢产物中的实用性。