An analysis strategy that can simultaneously determine the amino acid site information and configuration for polymyxin (PM) components is reported in this study. The main components of PMB1, PMB2, and PMB1-I (the Leucine at position 7 changed to be Isoleucine) were purified from the mixture of PMB. The PMB component was hydrolyzed by hydrochloric acid to be a mixture of amino acids, which were derivatized with Marfey’s reagent of N^α-(5-fluoro-2,4-dinitrophenyl)-l-alanine amide (l-FDAA), and then, the configurations of amino acids were easily determined by HPLC–MS. However, the aforementioned method could not offer the information of amino acid at each site, a specific enzymatic hydrolysis method combined Edman degradation was developed. The fatty acyl-diaminobutyric acid (position 1) of PMB component was removed by ficin, and the PMB components were transformed to be PMB nonapeptide. The amino acids at each site of the nonapeptide were determined by N-terminal sequencing. Combining the results of the above methods, the amino acid information and configuration of each site in PM components can be determined, especially the l-Leucine and l-Isoleucine in PMB1 and PMB1-I. The workflow was useful for other PM analogues to identify the sequence and configuration for quality control.

本研究报告了一种可以同时确定多粘菌素 (PM) 成分的氨基酸位点信息和配置的分析策略。从 PMB 混合物中纯化出 PMB1、PMB2 和 PMB1-I（第 7 位亮氨酸变为异亮氨酸）的主要成分。的PMB组分通过盐酸水解为氨基酸，将其衍生化的的Marfey试剂的混合物ñ ^α - （5-氟-2,4-二硝基苯基） -升-丙氨酸酰胺（升-FDAA)，然后，氨基酸的构型很容易通过 HPLC-MS 确定。然而，上述方法不能提供每个位点的氨基酸信息，开发了一种结合Edman降解的特异性酶水解方法。PMB组分的脂肪酰基二氨基丁酸（1位）被无花果苷去除，PMB组分转化为PMB九肽。九肽每个位点的氨基酸通过 N 端测序确定。结合上述方法的结果，可以确定PM组分中各位点的氨基酸信息和构型，尤其是l-亮氨酸和l-PMB1 和 PMB1-I 中的异亮氨酸。该工作流程对于其他 PM 类似物用于识别质量控制的序列和配置很有用。