Rahlwes Kathryn C, Puffal Julia, Morita Yasu S
Department of Microbiology, University of Massachusetts, Amherst, MA, USA.
Methods Mol Biol. 2019;1954:59-75. doi: 10.1007/978-1-4939-9154-9_6.
Mycobacteria and related bacteria in the Actinobacteria phylum are unusual in that they produce phosphatidylinositol (PI) as a major phospholipid species. PI can be further modified by glycan polymers, leading to the synthesis of PI mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). Small lipids such as PI and PIMs are extracted with a mixture of chloroform, methanol, and water and analyzed by thin layer chromatography. For larger glycolipids, such as LM and LAM, more hydrophilic solvent is needed for the extraction, and SDS-PAGE is better suited for the analysis. For LM, further structural characterization can be performed by MALDI-TOF mass spectrometry. Precise quantification of PIMs, LM, and LAM can be performed by quantification of glycan staining using analytical software. The metabolic radiolabeling protocol is also described.
放线菌门中的分枝杆菌及相关细菌与众不同，因为它们产生磷脂酰肌醇（PI）作为主要的磷脂种类。PI可被聚糖聚合物进一步修饰，从而导致合成PI甘露糖苷（PIM）、脂甘露聚糖（LM）和脂阿拉伯甘露聚糖（LAM）。诸如PI和PIM之类的小脂质用氯仿、甲醇和水的混合物提取，并通过薄层色谱法进行分析。对于较大的糖脂，如LM和LAM，则需要更具亲水性的溶剂进行提取，而SDS-PAGE更适合于分析。对于LM，可通过基质辅助激光解吸电离飞行时间质谱法进行进一步的结构表征。可使用分析软件通过聚糖染色定量对PIM、LM和LAM进行精确量化。还描述了代谢放射性标记方案。
