Quantitation of wall teichoic acid in Staphylococcus aureus by direct measurement of monomeric units using LC-MS/MS
Quantitation of wall teichoic acid in Staphylococcus aureus by direct measurement of monomeric units using LC-MS/MS
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DOI：
10.1016/j.ab.2016.10.027
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发表时间：
2017-02-01
影响因子：
2.9
通讯作者：
McLaren, David G.
中科院分区：
文献类型：
--
作者：
Berejnaia, Olga;Wang, Hao;McLaren, David G.
耐甲氧西林金黄色葡萄球菌（MRSA）的出现迫切需要能够应对这一威胁的新型治疗药物。我们之前曾报道过发现针对参与壁磷壁酸（WTA）生物合成的酶的新型抑制剂，并证明这些药物能够恢复β -内酰胺类药物对MRSA的疗效。在之前的那些报告中，通过聚丙烯酰胺凝胶电泳测量WTA水平的降低证明了抑制剂对相关途径的作用。为了能够对这些抑制剂进行更严格的分析，我们试图开发一种定量方法来测量WTA在全细胞中的减少量。在此，我们描述了一种稳健的方法，即通过液相色谱 - 串联质谱（LC - MS/MS）测量，将聚合态的WTA水解为单体成分核糖醇 - N - 乙酰氨基葡萄糖。研究发现该方案的关键要素包括氢氟酸介导的聚合态WTA水解的时间和温度，并且对这些参数的优化进行了详细描述。最重要的是，该检测方法能够准确且可重复地测量衣霉素以及新型TarO抑制剂（塔罗星类）代表物的半数有效浓度（EC50），以确定其对WTA的消耗情况。所描述的方法可以很容易地适用于定量感染小鼠模型组织匀浆中WTA的水平，突出了其在体外和体内特性研究中的适用性。（C）2016爱思唯尔公司。保留所有权利。
The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created an urgent need for new therapeutic agents capable of combating this threat. We have previously reported on the discovery of novel inhibitors targeting enzymes involved in the biosynthesis of wall teichoic acid (WTA) and demonstrated that these agents can restore beta-lactam efficacy against MRSA. In those previous reports pathway engagement of inhibitors was demonstrated by reduction in WTA levels measured by polyacrylamide gel electrophoresis. To enable a more rigorous analysis of these inhibitors we sought to develop a quantitative method for measuring whole-cell reductions in WTA. Herein we describe a robust methodology for hydrolyzing polymeric WTA to the monomeric component ribitol-N-acetylglucosamine coupled with measurement by LC-MS/MS. Critical elements of the protocol were found to include the time and temperature of hydrofluoric acid-mediated hydrolysis of polymeric WTA and optimization of these parameters is fully described. Most significantly, the assay enabled accurate and reproducible measurement of depletion EC50s for tunicamycin and representatives from the novel class of TarO inhibitors, the tarocins. The method described can readily be adapted to quantifying levels of WTA in tissue homogenates from a murine model of infection, highlighting the applicability for both in vitro and in vivo characterizations. (C) 2016 Elsevier Inc. All rights reserved.