Characterization of Lipid A Variants by Energy-Resolved Mass Spectrometry: Impact of Acyl Chains

Abstract

Lipid A molecules consist of a diglucosamine sugar core with a number of appended acyl chains which vary in their length and connectivity. Because of the challenging nature of characterizing these molecules and differentiating between isomeric species, an energy-resolved MS/MS strategy was undertaken to track the fragmentation trends and map genealogies of product ions originating from consecutive cleavages of acyl chains. Generalizations were developed based on the number and locations of the primary and secondary acyl chains as well as variations in preferential cleavages arising from the location of the phosphate groups. Secondary acyl chain cleavage occurs most readily for lipid A species at the 3′ position, followed by primary acyl chain fragmentation at both the 3′ and 3 positions. In the instances of bisphosphorylated lipid A variants, phosphate loss occurs readily in conjunction with the most favorable primary and secondary acyl chain cleavages.

Mapping phosphate modifications of substituted lipid Aviaa targeted MS3CID/UVPD strategy

“…This outcome is consistent with a previous report on energy variable mass spectrometry of lipid A molecules. 20 A single-stage UVPD method was also evaluated, and the outcome is shown in Figure S5 for the same PETN-modified lipid A described above. The UVPD spectrum produces some of the same glycosidic cleavage products as observed for CID/UVPD, such as the key 0,4 A 2 product ion which allows localization of the PETN on the reducing end of the molecule.…”

“…28,29 An alternative ion activation method, 193 nm ultraviolet photodissociation (UVPD), has proven to yield richer fragmentation patterns than conventional CID for lipid A, thus allowing additional structural details to be deciphered. 18–20,23,30–32 Specifically, UVPD cleaves the glycosidic (inter- and intra-ring) bonds of the lipid A backbone as well as promoting the characteristic neutral losses of acyl chains also typically observed upon collisional activation. 18,19 Further, CID and UVPD provide complementary information, with collisional activation typically producing fewer but more abundant fragment ions, and UVPD affording more details via unique diagnostic ions.…”

“…193 nm UVPD is used in the second stage of fragmentation to localize the substitutions, namely whether they are located at the 4′-phosphate position on the non-reducing end of the molecule or the 1-phosphate position on the reducing end of lipid A. Relative to the growing landscape of hybrid MS/MS and MS n strategies developed for lipids, 18–20,23,31,32 the current method provides several advances. The current CID-UVPD MS 3 approach affirms the identity of the substitutions based on mass confirmation.…”

Lipid A Remodeling Is a Pathoadaptive Mechanism That Impacts Lipopolysaccharide Recognition and Intracellular Survival of Burkholderia pseudomallei

“…Additional analysis of the fragmentation patterns can be accomplished when armed with the knowledge that only acyl chain substituted at the 3 or 3′ positions can be readily cleaved by MS in the negative mode ( 55 ). Furthermore, under constant power, the position 3-linked acyl chains will be cleaved at the lowest energy, followed by the position 3′-linked acyl chains ( 56 ). Both 1026b wild type and the pagL mutant have penta-acylated lipid A that can be fragmented by removal of the position 3-linked C 14:0 (3-OH).…”

Effect of matrices and additives on phosphorylated and ketodeoxyoctonic acid lipids A analysis by matrix‐assisted laser desorption ionization‐mass spectrometry

“…6,7 Different analytical techniques were used to elucidate the chemical structure and heterogeneity of lipid A found in numerous bacterial strains. Among them, mass spectrometry (MS) is one of the widely used techniques [8][9][10][11] ; particularly, matrix-assisted laser desorption ionization-time of flight MS (MALDI-ToF MS) was used for lipid analysis because of its speed and efficiency. Lipid A is substantially hydrophobic and is readily ionizable in the negative ion mode due to the terminal phosphate moieties.…”