Alvarez Laura, Cordier Baptiste, Van Teeffelen Sven, Cava Felipe
Department of Molecular Biology, Umeå University, Umeå, Sweden.
Microbial Morphogenesis and Growth Laboratory, Institut Pasteur, Paris, France.
Bio Protoc. 2020 Oct 5;10(19):e3780. doi: 10.21769/BioProtoc.3780.
Bacteria are surrounded by a protective peptidoglycan cell wall. Provided that this structure and the enzymes involved are the preferred target for our most successful antibiotics, determining its structural and chemical complexity is of the highest interest. Traditionally, high-performance liquid chromatography (HPLC) analyses have been performed, but these methods are very time consuming in terms of sample preparation and chromatographic separation. Here we describe an optimized method for preparation of Gram-negative bacteria peptidoglycan and its subsequent analysis by ultra-performance liquid chromatography (UPLC). The use of UPLC in peptidoglycan analyses provides a dramatic reduction of the sample volume and hands-on time required and, furthermore, permits in-line mass spectrometry (MS) of the UPLC resolved muropeptides, thus facilitating their identification. This method improves our capability to perform high throughput analysis to better understand the cell-wall biology.
细菌被一层保护性的肽聚糖细胞壁所包围。鉴于这种结构以及相关酶类是我们最有效的抗生素的首选作用靶点，确定其结构和化学复杂性具有至关重要的意义。传统上，人们采用高效液相色谱（HPLC）分析方法，但这些方法在样品制备和色谱分离方面非常耗时。在此，我们描述了一种用于制备革兰氏阴性菌肽聚糖并随后通过超高效液相色谱（UPLC）进行分析的优化方法。在肽聚糖分析中使用UPLC可显著减少所需的样品体积和实际操作时间，此外，还能对UPLC分离出的胞壁肽进行在线质谱（MS）分析，从而便于其鉴定。该方法提高了我们进行高通量分析以更好地理解细胞壁生物学的能力。
