Comprehensive Structure Characterization of Lipid A Extracted from Yersinia pestis for Determination of its Phosphorylation Configuration
Comprehensive Structure Characterization of Lipid A Extracted from Yersinia pestis for Determination of its Phosphorylation Configuration
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DOI：
10.1016/j.jasms.2010.01.008
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发表时间：
2010-05-01
影响因子：
3.2
通讯作者：
Ernst, Robert K.
中科院分区：
文献类型：
--
作者：
Jones, Jace W.;Cohen, Ilana E.;Ernst, Robert K.
我们报告的综合结构表征的脂质A提取鼠疫耶尔森菌（Yp）的磷酸化构型的测定，实现了结合分子生物学与高分辨率串联质谱的方法。最近已经发现从Yp提取的二磷酸化脂质A的磷酸化模式是C-1和C-4'二磷酸、C-1焦磷酸和C-4'焦磷酸的异质混合物（Proc.Natl. Acad. Sci. 2008，105，12742）。为了减少从Yp中提取的二磷酸化脂质A的固有磷酸异质性，我们将特异性C-1和C-4'位置磷酸酶掺入在37 ℃下生长的野生型K1 M6 + Yp中。从用C-1或C-4'磷酸酶修饰的Yp中提取的脂质A的全面高分辨率串联质谱分析允许单磷酸化和二磷酸化脂质A的明确结构分配以及异构的二磷酸盐和焦磷酸盐形式的区别。普遍的氨基阿拉伯糖修饰被确定为仅通过C-1和C-4'位置中的一个或两个位置处的磷酸二酯键与脂质A二糖连接。（J Am Soc Mass Spectrom 2010，21，785-799）（C）2010由Elsevier Inc.美国质谱学会American Society for Mass Spectrometry
We report on comprehensive structure characterization of lipid A extracted from Yersinia pestis (Yp) for determination of its phosphorylation configuration that was achieved by combining the methods of molecular biology with high-resolution tandem mass spectrometry. The phosphorylation pattern of diphosphorylated lipid A extracted from Yp has recently been found to be a heterogeneous mixture of C-1 and C-4' bisphosphate, C-1 pyrophosphate, and C-4' pyrophosphate (Proc. Natl. Acad. Sci. 2008, 105, 12742). To reduce the inherent phosphate heterogeneity of diphosphorylated lipid A extracted from Yp, we incorporated specific C-1 and C-4' position phosphatases into wild type K1M6+ Yp grown at 37 degrees C. Comprehensive high-resolution tandem mass spectrometric analyses of lipid A extracted from Yp modified with either the C-1 or C-4' phosphatase allowed for unambiguous structure assignment of monophosphorylated and diphosphorylated lipid A and distinction of isomeric bisphosphate and pyrophosphate forms. The prevalent aminoarabinose modification was determined to be exclusively attached to the lipid A disaccharide via a phospho-diester linkage at either or both the C-1 and C-4' positions. (J Am Soc Mass Spectrom 2010, 21, 785-799) (C) 2010 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry