Taxonomic group: bacteria / Proteobacteria
(Phylum: Proteobacteria)
Associated disease: infection due to Escherichia coli [ICD11:
XN6P4 
]
NCBI PubMed ID: 35293727Publication DOI: 10.1021/acs.analchem.1c04690Journal NLM ID: 0370536Correspondence: S. Nicolardi <s.nicolardi
lumc.nl>
Institutions: Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands, Janssen Vaccines AG (Branch of Cilag GmbH International), Rehhagstrasse 79, CH-3018 Bern, Switzerland, Bacterial Vaccine Discovery & Early Development, Janssen Vaccines and Prevention B.V., Leiden, The Netherlands
Bacterial glycoconjugate vaccines have a major role in preventing microbial infections. Immunogenic bacterial glycans, such as 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. Since development and quality control processes rely on such characterization, novel strategies are needed for faster and informative analysis. Here, we present a novel approach employing minimal sample preparation and ultrahigh-resolution mass spectrometry analysis for protein terminal sequencing and characterization of the oligosaccharide repeat units of bacterial glycoconjugate vaccines. Three glycoconjugate vaccine candidates, obtained from the bioconjugation of the O-antigen polysaccharides from E. coli serotypes O2, O6A, and O25B with the genetically detoxified exotoxin A from Pseudomonas 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 glycan fragments. 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 development.
O-antigen, Escherichia coli, mass spectrometry, glycoconjugate vaccine, FT-ICR MS, MALDI-in-source decay (ISD)
Structure type: polymer chemical repeating unit
Location inside paper: p. 4981, Fig. 2, EcoO6A
Compound class: O-polysaccharide, O-antigen
Contained glycoepitopes: IEDB_130648,IEDB_137340,IEDB_137473,IEDB_137485,IEDB_1391961,IEDB_141584,IEDB_141807,IEDB_142488,IEDB_144983,IEDB_146664,IEDB_151531,IEDB_152206,IEDB_885822,IEDB_983930,IEDB_983931,SB_192,SB_44,SB_72
Methods: MS/MS, bioconjugation, data analysis, MALDI-ISD FT-ICR MS
Related record ID(s): 8518, 21083
NCBI Taxonomy refs (TaxIDs): 217992Reference(s) to other database(s): GTC:G95910WE, GlycomeDB:
3565
Show glycosyltransferases
There is only one chemically distinct structure:
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