Altering Lipid A Precursor Ion Types in the Gas Phase for In-Depth Structural Elucidation via Tandem Mass Spectrometry

Lipid A, a well-known saccharolipid, acts as the inner lipid-glycan anchor of lipopolysaccharides in Gram-negative bacterial cell membranes and functions as an endotoxin. Its structure is composed of two glucosamines with β(1 → 6) linkages and various fatty acyl and phosphate groups. The lipid A structure can be used for the identification of bacterial species, but its complexity poses significant structural characterization challenges. In this work, we present a comprehensive strategy combining condensed-phase sample preparation, electrospray ionization, and gas-phase ion/ion reactions with tandem mass spectrometry for detailed lipid A structural elucidation. We use proton transfer reactions, charge-inversion reactions, and sequential ion/ion reactions for magnesium transfer to generate targeted lipid A ions. The strategy, established with a synthetic monophosphoryl lipid A (MPLA) and known MPLA and diphosphorylated lipid A (DPLA) from <i>Escherichia coli</i> F583, demonstrated that [MPLA - 2H]^2-, [MPLA - H]^-, and [MPLA - H + Mg]⁺ precursor ions offer complementary information for MPLA, while [DPLA - H]^-, [DPLA + H]⁺, and [DPLA - H + Mg]⁺ precursor ions provide analogous information for DPLA analysis. We validated the strategy using known lipid A species and also successfully applied this strategy to profile unknown MPLA and DPLA in the same <i>E. coli</i> strain.