Yang Hyojik, O'Keefe Ian, Smith Richard D, Sumner Kylie P, Sherman Matthew E, Goodlett David R, Sweet Charles R, Ernst Robert K
Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, Maryland 21201, United States.
Department of Pathology, School of Medicine, University of Maryland, Baltimore, Maryland 21201, United States.
Anal Chem. 2024 Aug 16;96(34):13838-45. doi: 10.1021/acs.analchem.4c01566.
Structural elucidation of Gram-negative bacterial lipid A traditionally requires chemical extraction followed by tandem MS data in the negative ion mode. Previously, we reported FLAT and FLAT as methods to rapidly determine the structure of lipid A without chromatographic techniques. In this work, we extend the capability and effectiveness of these techniques to elucidate the chemical structure in a de novo manner by including the use of positive ion mode (FLAT and FLAT) spectral approaches. Advantages of positive mode analysis of lipid A include the generation of more interpretable and informative fragmentation patterns that include the identification of diagnostic fragments, including selective dissociation of a glycosidic bond between two glucosamine units and the selective dissociation at the secondary acyl chain in 2'-N, allowing for the determination of the composition of fatty acids. As a proof of principle, we present here two previously uncharacterized structures of lipid A from () and (). In , we determined the lipid A structure with a nonconventional backbone of-β-1,6 linked 2,3-dideoxy-2,3-diamno-d-glucopyranose further modified with galacturonic acid in the place of typical 1-phosphate, and in , we assigned a single discrete structure using the specific fragmentation patterns of terminal phosphate groups present in lipid A. Therefore, FLAT, in combination with FLAT and FLAT, provides a multimodal structural platform for rapid structure characterization of unusual and complex lipid A structures from a single colony.
传统上，革兰氏阴性菌脂多糖A的结构解析需要先进行化学提取，然后在负离子模式下进行串联质谱分析。此前，我们报道了FLAT和FLAT方法，可在不使用色谱技术的情况下快速确定脂多糖A的结构。在这项工作中，我们通过采用正离子模式（FLAT和FLAT）光谱方法，扩展了这些技术从头解析化学结构的能力和有效性。脂多糖A正模式分析的优点包括生成更具可解释性和信息量的碎片模式，其中包括鉴定诊断性碎片，包括两个氨基葡萄糖单元之间糖苷键的选择性断裂以及2'-N位仲酰基链的选择性断裂，从而能够确定脂肪酸的组成。作为原理验证，我们在此展示了来自（）和（）的两种先前未表征的脂多糖A结构。在（）中，我们确定了一种具有非常规骨架的脂多糖A结构，其由β-1,6连接的2,3-二脱氧-2,3-二氨基-D-吡喃葡萄糖组成，典型的1-磷酸被半乳糖醛酸进一步修饰；在（）中，我们利用脂多糖A中存在的末端磷酸基团的特定碎片模式确定了单一离散结构。因此，FLAT与FLAT和FLAT相结合，为从单个菌落快速表征异常和复杂的脂多糖A结构提供了一个多模态结构平台。
