Structural Elucidation of Intact Rough-Type Lipopolysaccharides using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots.
Structural Elucidation of Intact Rough-Type Lipopolysaccharides using Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots.
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使用场不对称离子淌度谱和 Kendrick 质量缺陷图对完整粗糙型脂多糖进行结构解析。
DOI：
10.1101/2023.06.21.545950
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Goodlett,DavidR
中科院分区：
文献类型：
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作者：
Mikhael,Abanoub;Hardie,Darryl;Smith,Derek;Pětrošová,Helena;Ernst,RobertK;Goodlett,DavidR
脂多糖(LPS)是革兰氏阴性菌的标志性毒力因子。由于它们最简单的单位：脂肪酸和单糖的数量、类型和位置的不同，它们是复杂的、结构不同的混合物。因此，用传统的质谱学(MS)方法表征脂多糖的结构是具有挑战性的。在这里，我们描述了场不对称离子迁移率光谱(FAIMS)用于分析完整的R-型脂多糖复合体(LOS)的好处。使用广泛用于糖结合疫苗研究的TLR4激动剂Escherichia coliJ5(RC突变体)LOS进行结构表征。FAIMS气相分离提高了(S/N)的比值和所检测的LOS物种的数量。此外，FAIMS允许分离重叠的同素异位素，便于串联MS表征和明确的结构归属。除了FAIMS气相分馏的好处外，还利用Kendrick质量缺陷(KMD)图进一步完成了结构相关LOS分子的额外分选。值得注意的是，定制的KMD碱基单元[Na-H]创建了一个高度有序的KMD图，该图允许识别不同的LOS离子家族之间有趣和新颖的结构差异，即具有不同酰化度的离子、寡糖组成和化学修饰。用串联MS确定单个LOS离子的组成以及有组织的KMD图结构网络足以推断混合物中存在的321个LOS物种中的181个LOS物种的组成。FAIMS和KMD图的结合使人们能够深入描述复杂的LOS混合物，并揭示了关于其结构变化的大量新信息。
Lipopolysaccharides (LPSs) are a hallmark virulence factor of Gram-negative bacteria. They are complex, structurally heterogeneous mixtures due to variations in number, type, and position of their simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of an intact R-type lipopolysaccharide complex mixture (lipooligosaccharide; LOS). Structural characterization was performed usingEscherichia coliJ5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas-phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and unequivocal structural assignments. In addition to FAIMS gas-phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [Na–H] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families, i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 181 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations.