The focus of this review is the application of mass spectrometry to the structural characterization of bacterial lipopolysaccharides (LPSs), also referred to as "endotoxins," because they elicit the strong immune response in infected organisms. Recently, a wide variety of MS-based applications have been implemented to the structure elucidation of LPS. Methodological improvements, as well as on- and off-line separation procedures, proved the versatility of mass spectrometry to study complex LPS mixtures. Special attention is given in the review to the tandem mass spectrometric methods and protocols for the analyses of lipid A, the endotoxic principle of LPS. We compare and evaluate the different ionization techniques (MALDI, ESI) in view of their use in intact R- and S-type LPS and lipid A studies. Methods for sample preparation of LPS prior to mass spectrometric analysis are also described. The direct identification of intrinsic heterogeneities of most intact LPS and lipid A preparations is a particular challenge, for which separation techniques (e.g., TLC, slab-PAGE, CE, GC, HPLC) combined with mass spectrometry are often necessary. A brief summary of these combined methodologies to profile LPS molecular species is provided.

We established a new reversed phase-high performance liquid chromatography method combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross-reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups. In several cases, we observed the different retention of isobaric lipid A species, which had different secondary fatty acyl distribution at the C2' or the C3' sites. The fragmentation of the various, C4' monophosphorylated lipid A species in deprotonated forms provided structural assignment for each component. Fragmentation pathways of the tri-acylated, tetra-acylated, penta-acylated, hexa-acylated and hepta-acylated lipid A components and of the lipid A partial structures are suggested. As standards, the hexa-acylated ion at m/z 1716 with the E. coli-type acyl distribution and the hepta-acylated ion at m/z 1954 with the Salmonella-type acyl distribution were used. The results confirmed the presence of multiple forms of lipid A in all strains analyzed. In addition, the negative-ion mode MS permitted efficient detection for non-phosphorylated lipid A components, too. Copyright © 2016 John Wiley & Sons, Ltd.

Non-phosphorylated lipid A species confer reduced inflammatory potential for the bacteria. Knowledge on their chemical structure and presence in bacterial pathogens may contribute to the understanding of bacterial resistance and activation of the host innate immune system. In this study, we report the fragmentation pathways of negatively charged, non-phosphorylated lipid A species under low-energy collision-induced dissociation conditions of an electrospray ionization quadrupole time-of-flight instrument. Charge-promoted consecutive and competitive eliminations of the acyl chains and cross-ring cleavages of the sugar residues were observed. The A-type fragment ion series and the complementary X-type fragment(s) with corresponding deprotonated carboxamide(s) were diagnostic for the distribution of the primary and secondary acyl residues on the non-reducing and the reducing ends, respectively, of the non-phosphorylated lipid A backbone. Reversed-phase liquid chromatography in combination with negative-ion electrospray ionization quadrupole time-of-flight tandem mass spectrometry could provide sufficient information on the primary and secondary acyl residues of a non-phosphorylated lipid A. As a standard, the hexa-acylated ion at m/z 1636 with the Escherichia coli-type acyl distribution (from E. coli O111) was used. The method was tested and refined with the analysis of other non-phosphorylated hexa- and several hepta-, penta-, and tetra-acylated lipid A species detected in crude lipid A fractions from E. coli O111 and Proteus morganii O34 bacteria. Copyright © 2016 John Wiley & Sons, Ltd.

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In this study, we report the detailed analysis of the fragmentation patterns of positively charged lipid A species based on their tandem mass spectra obtained under low-energy collision-induced dissociation conditions of an electrospray quadrupole time-of-flight mass spectrometer. The tandem mass spectrometry experiments were performed after the separation of the compounds with a reversed-phase high performance liquid chromatography method. We found that both, phosphorylated and nonphosphorylated lipid A molecules can be readily ionized in the positive-ion mode by adduct formation with triethylamine added to the eluent. The tandem mass spectra of the lipid A triethylammonium adduct ions showed several product ions corresponding to inter-ring glycosidic cleavages of the sugar residues, as well as consecutive and competitive eliminations of fatty acids, phosphoric acid, and water following the neutral loss of triethylamine. Characteristic product ions provided direct information on the phosphorylation site(s), also when phosphorylation isomers (ie, containing either a C1 or a C4' phosphate group) were simultaneously present in the sample. Continuous series of high-abundance B-type and low-abundance Y-type inter-ring fragment ions were indicative of the fatty acyl distribution between the nonreducing and reducing ends of the lipid A backbone. The previously reported lipid A structures of Proteus morganii O34 and Escherichia coli O111 bacteria were used as standards. Although, the fragmentation pathways of the differently phosphorylated lipid A species significantly differed in the negative-ion mode, they were very similar in the positive-ion mode. The complementary use of positive-ion and negative-ion mode tandem mass spectrometry was found to be essential for the full structural characterization of the C1-monophosphorylated lipid A species.