Glycan and Protein Analysis of Glycoengineered Bacterial E. coli Vaccines by MALDI-in-Source Decay FT-ICR Mass Spectrometry

Abstract

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.

Carriers and Antigens: New Developments in Glycoconjugate Vaccines

“…Additionally, the conjugation rate, or quantity of polysaccharides conjugated to one single carrier protein, varies as well. For these reasons, the structure elucidation of the conjugate is not evident and requires several methods [ 72 , 73 ]. Next to this, there are some other challenges, such as OTases, that are not fully compatible with most bacterial O-SP, resulting in even more heterogeneous bioconjugates.…”

“…This new and exciting field of conjugate vaccines is already well studied ( Table 2 ). Nicolardi et al describe the development of a protein-based glycoengineered vaccine using the exotoxin A from P. aeruginosa as a carrier targeting E. coli serotypes O2, O6A and O25B [ 72 ]. Harding et al report (1) on a polyvalent pneumococcal bioconjugate vaccine using the natural acceptor protein ComP as a vaccine carrier decorated with S. pneumoniae CPSs [ 74 ] and (2) K. pneumoniae K1 and K2 serotypes produced in glycoengineered E. coli .…”

“…Both Nicolardi et al and Kowarik et al ( Figure 4 ) successfully constructed their bioconjugate via the traditional pathways and provide extensive data on the characterization. However, no immunogenicity data was provided making it impossible to assess the effectiveness [ 72 , 146 ]. Both Stevenson et al and Gening et al have provided successful immunogenicity data which was discussed for S. aureus already regarding the PNAG antigen and which could be applied towards E. coli as well [ 79 , 151 ] ( Table 13 ).…”

Direct MALDI Glycotyping of Glycoproteins toward Practical Subtyping of Biological Samples

“…16 We and Nicolardi et al recently reported the first example of glycan-selective MALDI−ISD analysis from intact glycoprotein. 17,18 A sodium-doped super DHB (DHB/5-methoxy salicylic acid/Na) caused random ISD fragmentation of artificial N-glycans having bacterial O-antigen polysaccharide repeats to give the sequence information of the repeat units. 18 A solid salt matrix composed of aniline/DHB/Na 19 caused selective ISD at the reducing ends of N-glycans and glycan fragment selective ionization, making this top-down glycomic analysis possible.…”

“…17 , 18 A sodium-doped super DHB (DHB/5-methoxy salicylic acid/Na) caused random ISD fragmentation of artificial N -glycans having bacterial O-antigen polysaccharide repeats to give the sequence information of the repeat units. 18 A solid salt matrix composed of aniline/DHB/Na 19 caused selective ISD at the reducing ends of N -glycans and glycan fragment selective ionization, making this top-down glycomic analysis possible. 17 However, the intensity of the glycan fragment ion signals obtained by MALDI–ISDMS analysis using this aniline/DHB/Na matrix was still insufficient to detect minor glycan components and to analyze glycoprotein containing biofluid.…”