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Mapping phosphate modifications of substituted lipid A via a targeted MS3 CID/UVPD strategy.

ABSTRACT: Structural characterization of lipid A from Gram-negative bacteria remains a significant challenge, especially with respect to localizing modifications of the phosphate groups typically found on the reducing and non-reducing ends of the ?-1',6-linked glucosamine disaccharide backbone of lipid A. As reported here, combining traditional collisional activated dissociation (CAD) and ultraviolet photodissociation (UVPD) in a hybrid MS3 approach facilitates identification and localization of substituents of the phosphate groups. The focus is on rapid identification and characterization of substituted lipid A species with specific emphasis on the modifications on the 1 and 4' phosphate moieties. Mapping these modifications, typically ones that modify the surface charges of lipopolysaccharides, is particularly important owing to the impact of these types of modifications on antibiotic resistance. The presence of phosphoethanolamine, aminoarabinose, and galactosamine moieties in hexaacylated and heptaacylated lipid A species, including ones from Enterobacter cloacae and Acinetobacter baumannii, are characterized using a targeted MS3 strategy to identify glycosidic product ions (1,5X1 and 0,4A2, typically) which allow localization of the substituents.

SUBMITTER: Crittenden CM

PROVIDER: S-EPMC6019210 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Mapping phosphate modifications of substituted lipid A via a targeted MS<sup>3</sup> CID/UVPD strategy.

Crittenden Christopher M CM Herrera Carmen M CM Williams Peggy E PE Ricci Dante P DP Swem Lee R LR Trent M Stephen MS Brodbelt Jennifer S JS

The Analyst 20180601 13

Structural characterization of lipid A from Gram-negative bacteria remains a significant challenge, especially with respect to localizing modifications of the phosphate groups typically found on the reducing and non-reducing ends of the β-1',6-linked glucosamine disaccharide backbone of lipid A. As reported here, combining traditional collisional activated dissociation (CAD) and ultraviolet photodissociation (UVPD) in a hybrid MS3 approach facilitates identification and localization of substitue ...[more]

PMID: 29881855

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Project description:DNA can be damaged through covalent modifications of the nucleobases by endogenous processes. These modifications, commonly referred to as DNA adducts, can persist and may lead to mutations, and ultimately to the initiation of cancer. A screening methodology for the majority of known endogenous DNA adducts would be a powerful tool for investigating the etiology of cancer and for the identification of individuals at high-risk to the detrimental effects of DNA damage. This idea led to the development of a DNA adductomic approach using high resolution data-dependent scanning, an extensive MS2 fragmentation inclusion list of known endogenous adducts, and neutral loss MS3 triggering to profile all DNA modifications. In this method, the detection of endogenous DNA adducts is performed by observation of their corresponding MS3 neutral loss triggered events and their relative quantitation using the corresponding full scan extracted ion chromatograms. The method's inclusion list consists of the majority of known endogenous DNA adducts, compiled, and reported here, as well as adducts specific to tobacco exposure included to compare the performance of the method with previously developed targeted approaches. The sensitivity of the method was maximized by reduction of extraneous background signal through the purification and minimization of the amount of commercially obtained enzymes used for the DNA hydrolysis. In addition, post-hydrolysis sample purification was performed using off-line HPLC fraction collection to eliminate the highly abundant unmodified bases, and to avoid introduction of plasticizers found in solid-phase extraction cartridges. Also, several instrument parameters were evaluated to optimize the ion signal intensities and fragmentation spectra quality. The method was tested on an animal model of lung carcinogenesis where A/J mice were exposed to the tobacco specific lung carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) with its effects enhanced by co-exposure to the pro-inflammatory agent lipopolysaccharide (LPS). Lung DNA were screened for endogenous DNA adducts known to result from oxidative stress and LPS-induced lipid peroxidation, as well as for adducts due to NNK exposure. The relative quantitation of the detected DNA adducts was performed using parallel reaction monitoring MS2 analysis, demonstrating a general workflow for analysis of endogenous DNA adducts.

Project description: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.

Dataset Information

Mapping phosphate modifications of substituted lipid A via a targeted MS3 CID/UVPD strategy.

Publications

Mapping phosphate modifications of substituted lipid A via a targeted MS<sup>3</sup> CID/UVPD strategy.

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