Stankeviciute Gabriele, Klein Eric A
Center for Computational and Integrative Biology, Rutgers University-Camden, Camden, NJ, 08102, USA.
Biology Department, Rutgers University-Camden, Camden, NJ, 08102, USA.
Bio Protoc. 2019 Nov 5;9(21):e3421. doi: 10.21769/BioProtoc.3421.
The peptidoglycan sacculus, or cell wall, is what defines bacterial cell shape. Cell wall composition can be best characterized at the molecular level by digesting the peptidoglycan murein polymer into its muropeptide subunits and quantifying the abundance of muropeptides using high-pressure liquid chromatography. Certain features of the cell wall including muropeptide composition, glycan strand length, degree of crosslinking, type of crosslinking and other peptidoglycan modifications can be quantified using this approach. Well-established protocols provide us with highly-resolved and quantitatively reproducible chromatographic data, which can be used to investigate bacterial cell wall composition under a variety of environmental or genetic perturbations. The method described here enables the purification of muropeptide samples, their quantification by HPLC, and fraction collection for peak identification by mass spectrometry. Although the methods for peptidoglycan purification and HPLC analysis have been previously published, our method includes important details on how to re-equilibrate the column between runs to allow for automated analysis of multiple samples.
肽聚糖囊泡，即细胞壁，决定了细菌的细胞形状。通过将肽聚糖胞壁质聚合物消化成其胞壁肽亚基，并使用高压液相色谱法定量胞壁肽的丰度，可在分子水平上最好地表征细胞壁的组成。使用这种方法可以量化细胞壁的某些特征，包括胞壁肽组成、聚糖链长度、交联程度、交联类型和其他肽聚糖修饰。成熟的方案为我们提供了高分辨率且可定量重现的色谱数据，可用于研究各种环境或遗传扰动下的细菌细胞壁组成。这里描述的方法能够纯化胞壁肽样品，通过高效液相色谱法对其进行定量，并收集馏分以通过质谱法进行峰鉴定。尽管肽聚糖纯化和高效液相色谱分析方法此前已发表，但我们的方法包含了关于如何在运行之间重新平衡色谱柱以实现多个样品自动分析的重要细节。
