Mass Spectrometry Laboratory, MolSys Research Unit, Quartier Agora, University of Liège, Allée du Six Août 11, B-4000 Liège, Belgium.
Centre for Protein Engineering, InBioS Research Unit, Quartier Agora, University of Liège, Allée du Six Août 13, B-4000 Liège, Belgium.
Anal Chem. 2021 Feb 2;93(4):2342-2350. doi: 10.1021/acs.analchem.0c04218. Epub 2021 Jan 20.
Peptidoglycan (PGN) is an essential structure found in the bacterial cell wall. During the bacterial life cycle, PGN continuously undergoes biosynthesis and degradation to ensure bacterial growth and division. The resulting PGN fragments (muropeptides and peptides), which are generated by the bacterial autolytic system, are usually transported into the cytoplasm to be recycled. On the other hand, PGN fragments can act as messenger molecules involved in the bacterial cell wall stress response as in the case of β-lactamase induction in the presence of β-lactam antibiotic or in triggering mammalian innate immune response. During their cellular life, bacteria modulate their PGN degradation by their autolytic system or their recognition by the mammalian innate immune system by chemically modifying their PGN. Among these modifications, the amidation of the ε-carboxyl group of -diaminopimelic acid present in the PGN peptide chain is frequently observed. Currently, the detection and quantitation of PGN-derived peptides is still challenging because of the difficulty in separating these highly hydrophilic molecules by RP-HPLC as these compounds are eluted closely after the column void volume or coeluted in many cases. Here, we report the use of capillary zone electrophoresis coupled via an electrospray-based CE-MS interface to high-resolution mass spectrometry for the quantitation of three PGN peptides of interest and their amidated derivatives in bacterial cytoplasmic extracts. The absolute quantitation of the tripeptide based on the [C,N] isotopically labeled standard was also performed in crude cytoplasmic extracts of bacteria grown in the presence or absence of a β-lactam antibiotic (cephalosporin C). Despite the high complexity of the samples, the repeatability of the CZE-MS quantitation results was excellent, with relative standard deviations close to 1%. The global reproducibility of the method including biological handling was better than 20%.
肽聚糖（PGN）是细菌细胞壁中存在的一种重要结构。在细菌的生命周期中，PGN 不断进行生物合成和降解，以确保细菌的生长和分裂。由细菌自溶系统产生的 PGN 片段（肽聚糖和肽）通常被转运到细胞质中进行回收。另一方面，PGN 片段可以作为信使分子参与细菌细胞壁应激反应，例如在β-内酰胺抗生素存在下诱导β-内酰胺酶，或触发哺乳动物先天免疫反应。在它们的细胞生命中，细菌通过其自溶系统或其被哺乳动物先天免疫系统识别来调节其 PGN 降解，通过化学修饰其 PGN。在这些修饰中，经常观察到存在于 PGN 肽链中的 -二氨基庚二酸的 ε-羧基的酰胺化。目前，由于这些高度亲水性分子通过反相高效液相色谱法（RP-HPLC）分离非常困难，因为这些化合物在柱空隙体积洗脱后紧密洗脱或在许多情况下共洗脱，因此仍然难以检测和定量 PGN 衍生肽。在这里，我们报告了使用毛细管区带电泳通过基于电喷雾的 CE-MS 接口与高分辨率质谱联用，对细菌细胞质提取物中三种感兴趣的 PGN 肽及其酰胺化衍生物进行定量。还在存在或不存在β-内酰胺抗生素（头孢菌素 C）的情况下生长的细菌的粗细胞质提取物中基于 [C，N] 同位素标记标准对三肽进行了绝对定量。尽管样品的复杂性很高，但 CZE-MS 定量结果的可重复性非常好，相对标准偏差接近 1%。包括生物学处理在内的方法的总体重现性优于 20%。
