Abstract
Lipid A, the membrane-bound phosphoglycolipid component of bacteria, is held responsible for the clinical syndrome of gram-negative sepsis. In this study, the fragmentation behavior of a set of synthetic lipid A derivatives was studied by electrospray ionization multistage mass spectrometry (ESI-MSn), in conjunction with tandem mass spectrometry (MS/MS), using low-energy collision-induced dissociation (CID). Genealogical insight about the fragmentation pathways of the deprotonated 4’-monophosphoryl lipid A structural analogs led to proposals of a number of alternative dissociation routes that have not been reported previously. Each of the fragment ions was interpreted using various possible mechanisms, consistent with the principles of reactions described in organic chemistry. Specifically, the hypothesized mechanisms are: (i) cleavage of the C-3 primary fatty acid leaves behind an epoxide group attached to the reducing sugar; (ii) cleavage of the C-3’ primary fatty acid (as an acid) generates a cyclic phosphate connected to the nonreducing sugar; (iii) cleavage of the C-2’ secondary fatty acid occurs both in acid and ketene forms; iv) the C-2 and C-2’ primary fatty acids are eliminated as an amide and ketene, respectively; (v) the 0,2A2 cross-ring fragment contains a four-membered ring (oxetanose); (vi) the 0,4A2 ion is consecutively formed from the 0,2A2 ion by retro-aldol, retro-cycloaddition, and transesterification; and (vii) formations of H2PO4− and PO3− are associated with the formation of sugar epoxide. An understanding of the relation between 0,2A2 and 0,4A2-type sugar fragments and the different cleavage mechanisms of the two ester-linked primary fatty acids is invaluable for distinguishing lipid A isomers with different locations of a single ester-linked fatty acid (i.e., at C-3 or C-3’). Thus, in addition to a better comprehension of lipid A fragmentation processes in mass spectrometers, our observations can be applied for a more precise elucidation of naturally occurring lipid A structures.
Highlights
Lipid A is the active component of lipopolysaccharides found in the outer membrane of most gram-negative bacteria [1]
One major peak was detected in the mass spectrum, corresponding to the deprotonated 4’-monophosphoryl lipid A molecule [M − H]−
Fragmentation patterns of each standard were studied at the MS2 stage, and results obtained with the two mass spectrometers at different RF amplitudes or collision energies (QqQ), respectively, were compared
Summary
Lipid A is the active component of lipopolysaccharides ( called endotoxins) found in the outer membrane of most gram-negative bacteria [1]. On the other hand, depending on the dose and the structure of lipid A, the resulting immune response can lead to the uncontrolled production of inflammatory cytokines that may evoke fatal effects, such as high fever, septic shock, and multiorgan failure [2]. In this context, it is not surprising that several lipid A derivatives (synthetic or produced from bacteria) have been targets for developing effective adjuvants in vaccines and immunotherapy formulations [3,4]. Alterations in the acylation and/or phosphorylation pattern, as well as post-translational modifications, significantly influence the immune system recognition (or evasion) [7] and potency of response [8] and may even modulate bacterial resistance to antibiotics [9]
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