Glycan and protein analysis of glycoengineered bacterial E. coli vaccines by MALDI-in-source decay FT-ICR mass spectrometry

Bacterial glycoconjugate vaccines have a major role in preventing microbial infections. Immunogenic bacterial glycans, suchas O-antigen polysaccharides, can be recombinantly expressed and combined with specific carrier proteins to produce effective vaccines. O-Antigen polysaccharides are typically polydisperse, and carrier proteins can have multiple glycosylation sites. Consequently, recombinant glycoconjugate vaccines have a high structural heterogeneity, making their characterization challenging. Sincedevelopment and quality control processes rely on such character-ization, novel strategies are needed for faster and informative analysis.Here, we present a novel approach employing minimal samplepreparation and ultrahigh-resolution mass spectrometry analysis forprotein terminal sequencing and characterization of the oligosaccharide repeat units of bacterial glycoconjugate vaccines. Threeglycoconjugate vaccine candidates, obtained from the bioconjugation of the O-antigen polysaccharides fromE. coliserotypes O2,O6A, and O25B with the genetically detoxified exotoxin A fromPseudomonas aeruginosa, were analyzed by MALDI-in-source decay(ISD) FT-ICR MS. Protein and glycan ISD fragment ions were selectively detected using 1,5-diaminonaphtalene and a 2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxybenzoic acid mixture (super-DHB) as a MALDI matrix, respectively. The analysis of protein fragments required the absence of salts in the samples, while the presence of salt was key for the detection of sodiated glycanfragments. MS/MS analysis of O-antigen ISD fragments allowed for the detection of specific repeat unit signatures. The developed strategy requires minute sample amounts, avoids the use of chemical derivatizations, and comes with minimal hands-on time allowing for fast corroboration of key structural features of bacterial glycoconjugate vaccines during early- and late-stage developmentProteomic

Capsular polysaccharide from <i>Klebsiella</i> K19. Part II : Depolymerization of capsular polysaccharide from <i>Klebsiella</i> K19 the capsular polysaccharide from <i>Klebsiella</i> K19 by the glycanase associated with particles of <i>Klebsiella</i> Becteriophage phi19

International audienc

Capsular polysaccharide from <i>Klebsiella</i> K19. Part I : Structural investigation of the capsular polysaccharide from <i>Klebsiella</i> K19 by chemical and NMR analysis

International audienc

Structure and immunochemistry of meningococcal lipopolysaccharides

NRC publication: Ye

Homologous and heterologous reactions of bacteriophages phi41 and phi12 on capsular polysaccharides from <i>Klebsiella</i> K41 and K12

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Structure and immunochemisry of meningococcal lipopolysaccharides

NRC publication: Ye

Proof of the occurence of 5,6-O-(1-carboxyethylidene)-D-galactofuranose units in the capsular polysaccharide of <i>Klebsiella</i> K12

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Structure of the L5 lipopolysaccharide core oligosaccharides of Neisseria meningitidis

Three different oligosaccharides were isolated by mild acid hydrolysis of the lipopolysaccharides, obtained from Neisseria meningitidis serotype 5, and their structures were elucidated by combined chemical and physical techniques. The use of 500-MHz 1H NMR in both one-dimensional and two-dimensional modes as well as nuclear Overhauser effect experiments were employed. To assist in the structural assignments the purified oligosaccharides were also degraded by chemical and enzymatic procedures to smaller fragments. The largest of the three original oligosaccharides is a triantennary partially O-acetylated decasaccharide in which the largest antenna terminates in a lacto-N-neotetraose unit. The smaller oligosaccharides (heptasaccharide and octasaccharide) except for terminal glycose deletions from the longest antenna are structural replicas of the largerPeer reviewed: YesNRC publication: Ye