193 nm Ultraviolet Photodissociation Mass Spectrometry for the Structural Elucidation of Lipid A Compounds in Complex Mixtures

Abstract

Here we implement ultraviolet photodissociation (UVPD) in an online liquid chromatographic tandem mass spectrometry (MS/MS) strategy to support analysis of complex mixtures of lipid A combinatorially modified during development of vaccine adjuvants. UVPD mass spectrometry at 193 nm was utilized to characterize the structures and fragment ion types of lipid A from Escherichia coli, Vibrio cholerae, and Pseudomonas aeruginosa using an Orbitrap mass spectrometer. The fragment ions generated by UVPD were compared to those from collision induced dissociation (CID) and higher energy collision dissociation (HCD) with respect to the precursor charge state. UVPD afforded the widest array of fragment ion types including acyl chain C–O, C–N, and C–C bond cleavages and glycosidic C–O and cross ring cleavages, thus providing the most comprehensive structural analysis of the lipid A. UVPD exhibited virtually no dependence on precursor ion charge state and was best at determining lipid A structure including acyl chain length and composition, giving it an advantage over collision based methods. UVPD was incorporated into an LC–MS/MS methodology for the analysis of a number of structural variants in a complex mixture of combinatorially engineered Escherichia coli lipid A.

Collision-induced dissociation of doubly-charged barium-cationized lipids generated from liquid samples by atmospheric pressure matrix-assisted laser desorption/ionization provides structurally diagnostic product ions

“…Ultraviolet photodissociation (UVPD) is another promising approach for lipid characterization [32,33]. UVPD has recently been implemented on commercial instrumentation, initially requiring significant non-trivial instrument modifications [34,35].…”

Top-down strategies for the structural elucidation of intact gram-negative bacterial endotoxins

“…As a promising alternative, UVPD is a higher energy activation method that has demonstrated advantages over CID for characterization of a wide range of biopolymers (including lipids, proteins, peptides, nucleic acids, and glycans). 41,43–66 Hybrid activation methods consisting of UVPD in conjunction with CID or electron transfer dissociation (ETD) have also been developed as a means to yield even richer fragmentation patterns. 44,46 We have explored UVPD at 193 nm for characterization of glycolipids, including lipid A, and found that UV photoexcitation promotes extensive C-C, C-N, C-O, glycosidic and cross-ring cleavages which contribute to the highly informative fragmentation patterns observed upon UVPD.…”

“…44,46 We have explored UVPD at 193 nm for characterization of glycolipids, including lipid A, and found that UV photoexcitation promotes extensive C-C, C-N, C-O, glycosidic and cross-ring cleavages which contribute to the highly informative fragmentation patterns observed upon UVPD. 41,61–66 UVPD has allowed unusual modifications to be accurately pinpointed and isobaric species to be differentiated. 41,61–66 …”

“…41,61–66 UVPD has allowed unusual modifications to be accurately pinpointed and isobaric species to be differentiated. 41,61–66 …”

Re-print of “Radical Delivery and Fragmentation for Structural Analysis of Glycerophospholipids”

“…The radical species generated by PD can be collisionally activated to stimulate migration of the radical that eventually leads to radical directed dissociation (RDD). This method differs from other photoactivation experiments where photons are the primary initiators of the observed fragmentation, as is the case for ultraviolet photodissociation (UVPD) [26][27][28][29] and infrared multi-photon dissociation (IRMPD) [30,31] based experiments.…”