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Detailed structural characterization of intact rough-type lipopolysaccharides (R-LPS) was accomplished using a multi-stage mass spectrometry (MS(3)) strategy consisting of collision-induced dissociation (CID) followed by 193 ultraviolet photodissociation (UVPD) implemented on an Orbitrap Fusion mass spectrometer. Complex mixtures of R-LPS from either Escherichia coli or Salmonella enterica were directly infused into the mass spectrometer using static source nanoelectrospray ionization (nanoESI). An initial CID event performed on an R-LPS precursor produced spectra with abundant ions corresponding to the lipid A and core oligosaccharide (OS) substructures. Comparison of CID spectra of R-LPS ions with varying lipid A and core OS structures verifies that lipid A and core OS ions are consistently produced in high abundance. The resulting lipid A and core OS ions were subsequently activated by CID, high-energy collision-induced dissociation (HCD), or UVPD. For both the lipid A and core OS substructures, HCD and UVPD produced highly informative complementary spectra, with UVPD of the core OS producing an extensive array of cross-ring cleavage fragments. Successful discernment of E. coli R-LPS structures with isomeric core structures confirmed the degree to which subtle structural differences could be determined using this method.

Shotgun Analysis of Rough-Type Lipopolysaccharides Using Ultraviolet Photodissociation Mass Spectrometry