Ultra-Sensitive, High-Resolution Liquid Chromatography Methods for the High-Throughput Quantitative Analysis of Bacterial Cell Wall Chemistry and Structure
Ultra-Sensitive, High-Resolution Liquid Chromatography Methods for the High-Throughput Quantitative Analysis of Bacterial Cell Wall Chemistry and Structure
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DOI：
10.1007/978-1-4939-3676-2_2
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发表时间：
2016-01-01
期刊：
影响因子：
--
通讯作者：
Cava, Felipe
中科院分区：
文献类型：
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作者：
Alvarez, Laura;Hernandez, Sara B.;Cava, Felipe
高效液相色谱（HPLC）分析已成为确定细胞壁结构和化学复杂性的关键。然而，这种方法在样品制备和色谱分离方面非常耗时。本文介绍了(1)从革兰氏阴性菌和革兰氏阳性菌中分离肽聚糖的优化方法，该方法大大缩短了样品制备时间；(2)快速高效的超高效液相色谱（UPLC）技术在多肽分离和定量中的应用。分析仪器和固定相化学的进步已经允许改进的方案，将运行时间从小时（2-3小时）缩短到分钟（10-20分钟），并将样品需求减少至少一个数量级。此外，基于有机溶剂的方法的发展允许在线质谱（MS）对uplc分解的多肽进行分析。将这些技术应用于高通量分析将加快对细胞壁生物学的更好理解。
High-performance liquid chromatography (HPLC) analysis has been critical for determining the structural and chemical complexity of the cell wall. However this method is very time consuming in terms of sample preparation and chromatographic separation. Here we describe (1) optimized methods for peptidoglycan isolation from both Gram-negative and Gram-positive bacteria that dramatically reduce the sample preparation time, and (2) the application of the fast and highly efficient ultra-performance liquid chromatography (UPLC) technology to muropeptide separation and quantification. The advances in both analytical instrumentation and stationary-phase chemistry have allowed for evolved protocols which cut run time from hours (2-3 h) to minutes (10-20 min), and sample demands by at least one order of magnitude. Furthermore, development of methods based on organic solvents permits in-line mass spectrometry (MS) of the UPLC-resolved muropeptides. Application of these technologies to high-throughput analysis will expedite the better understanding of the cell wall biology.