C. difficile strain 630 flagellin C modifications

In proteomics, the study of post-translational modifications often relies on some type of enrichment strategy. Here, we show that an approach that is commonly used in phosphoproteomics (Immobilised Metal Affinity Chromatography (Fe3+-IMAC)) also enriches for peptides with a unique post-translational modification, known as type A, in the human pathogen Clostridioides difficile. The type A modification consists of a monosaccharide (GlcNAc) that is linked to an N-methylated threonine through a phosphodiester bond. This structure has previously been described on flagellin C of several C. difficile strains and is important for bacterial motility. Using LC-MS/MS analyses of IMAC-captured tryptic peptides, we not only observed Type A modified C. difficile flagellin peptides but also a variety of truncated/modified Type A structures on these peptides. Using an elaborate set of mass spectrometry analyses, we demonstrate that one of these modifications consists of a Type A structure containing a phosphonate (2-aminoethylphosphonate, 2-AEP), a modification that is rarely observed and has hitherto not been described in C. difficile.

在蛋白质组学（proteomics）研究中，翻译后修饰的解析通常依赖于各类富集策略。本研究证实，磷酸化蛋白质组学（phosphoproteomics）中常用的固定金属亲和色谱（Immobilised Metal Affinity Chromatography，Fe³⁺-IMAC）策略，同样可富集人类致病菌艰难梭菌（Clostridioides difficile）中携带独特翻译后修饰的肽段，该修饰被命名为A型修饰。A型修饰的结构为：单糖（GlcNAc）通过磷酸二酯键连接至N-甲基化苏氨酸。此前已有研究在多种艰难梭菌菌株的鞭毛蛋白C（flagellin C）上报道了该结构，且其对细菌运动性具有关键作用。通过对固定金属亲和色谱捕获的胰酶解肽进行液相色谱-串联质谱（LC-MS/MS）分析，我们不仅检测到了携带A型修饰的艰难梭菌鞭毛蛋白肽段，还在这类肽段上发现了多种截短/修饰后的A型变体结构。借助一套系统的质谱分析手段，我们证实其中一种修饰为携带膦酸酯（phosphonate）的A型结构，具体为2-氨基乙基膦酸（2-aminoethylphosphonate，2-AEP）——该修饰极为罕见，此前从未在艰难梭菌中被报道过。