Benefits of Field Asymmetric Ion Mobility Spectrometry and Kendrick Mass Defect Plots in Lipid A Analysis

Lipid A, the endotoxic component of Gram-negative bacterial lipopolysaccharide (LPS), consists of a phosphorylated glucosamine disaccharide backbone with variable fatty acyl chains. It is central to bacterial virulence and immune activation, and its structural diversity is best characterized by electrospray ionization tandem mass spectrometry (ESI-MS/MS). However, conventional shotgun ESI-MS analysis, while rapid, often produces highly congested spectra in which isobaric species and undetected aggregates overlap, limiting confident structural assignment and slowing data interpretation. Despite this, ion mobility-based approaches such as field asymmetric ion mobility spectrometry (FAIMS) have not been systematically applied to typical lipid A mixtures, and their ability to resolve lipid A aggregates remains largely unexplored. Here, we describe the benefits of coupling FAIMS with ESI-MS/MS for lipid A analysis. Additionally, classifying and sorting chemically related lipid A molecules by Kendrick mass defect (KMD) plots using a fractional base unit are presented. Using <i>Aeromonas hydrophila</i> (<i>A. hydrophila</i> serovar SJ-26) lipid A, FAIMS allowed the fractionation of lipid A ions into three major classes: diphosphorylated lipid A (DPLA), monophosphorylated lipid A (MPLA), and unexpected noncovalent MPLA dimers. Major precursor ions of each class were elucidated by tandem MS. Additionally, the heterogeneity in each lipid A fraction was investigated using KMD plots. Integrating FAIMS separation, tandem MS, and KMD analysis enabled detailed lipid A analysis. Importantly, this analysis revealed the presence of unexpected aggregate dimers that could easily confound mass spectrometry interpretation as well as biological activity assays, including TLR4 activation and cytokine induction.