Recent advances in lipopolysaccharide (LPS) biology have led to its use in drug discovery pipelines, including vaccine and vaccine adjuvant discovery. Desirable characteristics for LPS vaccine candidates include both the ability to produce a specific antibody titer in patients and a minimal host inflammatory response directed by the innate immune system. However, in-depth chemical characterization of most LPS extracts has not been performed; hence, biological activities of these extracts are unpredictable. Additionally, the most widely adopted workflow for LPS structure elucidation includes nonspecific chemical decomposition steps before analyses, making structures inferred and not necessarily biologically relevant. In this work, several different mass spectrometry workflows that have not been previously explored were employed to show proof-of-principle for top down LPS primary structure elucidation, specifically for a rough-type mutant (J5) E. coli-derived LPS component of a vaccine candidate. First, ion mobility filtered precursor ions were subjected to collision induced dissociation (CID) to define differences in native J5 LPS v. chemically detoxified J5 LPS (dLPS). Next, ultra-high mass resolving power, accurate mass spectrometry was employed for unequivocal precursor and product ion empirical formulae generation. Finally, MS³ analyses in an ion trap instrument showed that previous knowledge about dissociation of LPS components can be used to reconstruct and sequence LPS in a top down fashion. A structural rationale is also explained for differential inflammatory dose-response curves, in vitro, when HEK-Blue hTLR4 cells were administered increasing concentrations of native J5 LPS v. dLPS, which will be useful in future drug discovery efforts.

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脂多糖（LPS）生物学的最新进展已导致其在药物发现管道中的使用，包括疫苗和疫苗佐剂发现。LPS候选疫苗的理想特性包括在患者体内产生特定抗体滴度的能力以及由先天免疫系统指导的最小的宿主炎症反应。但是，尚未对大多数LPS提取物进行深入的化学表征。因此，这些提取物的生物活性是不可预测的。此外，用于LPS结构阐明的最广泛采用的工作流程包括在分析之前进行非特异性化学分解的步骤，从而推断出结构，并不一定具有生物学意义。在这项工作中，大肠杆菌候选疫苗的LPS成分。首先，对经过离子淌度过滤的母离子进行碰撞诱导解离（CID），以定义天然J5 LPS与化学解毒的J5 LPS（dLPS）的差异。接下来，采用超高质量的分辨能力，精确的质谱分析方法来生成明确的前体和产物离子经验公式。最后，MS ³在离子阱仪器中进行的分析表明，以前有关LPS组分解离的知识可用于以自上而下的方式重建LPS并对其进行测序。在体外，当给予HEK-Blue hTLR4细胞增加浓度的天然J5 LPS v.dLPS浓度时，还可以解释差异炎症剂量-反应曲线的结构原理，这将在将来的药物开发工作中有用。

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