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, 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.


S2
MALDI-ISD FT-ICR mass spectra of glycoconjugate EcoO25B acquired in positive and negative ion modes S3

Figure S2
Enlargements of the MALDI-ISD FT-ICR mass spectrum of glycoconjugate EcoO25B depicted in Figure S1A S4

Figure S3
Examples of fragment ions detected in positive and negative ion modes S5 Figure S4 Enlargements of negative mode MALDI-ISD FT-ICR mass spectra of the glycoconjugates EcoO2, EcoO6A, and EcoO25B S7

Figure S5
Enlargements of negative mode MALDI-ISD FT-ICR mass spectra of the glycoconjugates EcoO2, EcoO6A, and EcoO25B S7 Figure S6 Schematic representation of the standardized nomenclature of fragment ions of carbohydrates S8

Figure S7
Enlargement of the MALDI-ISD FT-ICR mass spectra of the glycoconjugates EcoO2, EcoO6A, and EcoO25B in the m/z-range 3000-8000 S9

Figure S8
Enlargement of the MALDI-ISD FT-ICR mass spectrum of the glycoconjugate EcoO6A in the m/z-range 4000-12000 S10

Figure S9
Enlargement of the MALDI-ISD FT-ICR mass spectrum of the glycoconjugate EcoO6A in the m/z-range between 1RU and 2RU S11

Figure S10
Enlargement of the MALDI-ISD FT-ICR mass spectrum of the glycoconjugate EcoO25B in the m/z-range between 1RU and 2RU S12 Figure S1. Examples of MALDI-ISD FT-ICR mass spectra acquired in (A) positive and (B) negative ion modes after desalting of the glycoconjugate EcoO25B. Such spectra were visually investigated and the signals matching the theoretical m/z-values of ISD fragment ions of EPA protein were assigned (see Figure S2). The lists of the assigned fragment ions are reported in Table S4. Enlargements of the spectrum depicted in A are reported in Figures S2. The spectrum depicted in B is characterized by the presence of intense MALDI ion clusters that are typically detected below m/z 1000. Ultrahighresolution measurements allowed for confident assignment in this region. Figure S2. Enlargements of the MALDI-ISD FT-ICR mass spectrum of glycoconjugate EcoO25B depicted in Figure S1A. Detected c'-, yand z'-type fragment ions were compared with theoretical values and assigned within a mass measurement error of 15 ppm. The assigned fragments are reported in Table S4. * indicates z'+1,5-DAN ions. Blue circle indicates w-type ions.
S5 Figure S2. Continued Figure S3. Examples of fragment ions detected in positive and negative ion modes. As previously shown (Nicolardi et al., Anal. Chem. 2020, 92, 18, 12429-12436), negative mode MALDI-ISD FT-ICR MS can provide a higher signal intensity of fragment ions in the m/z-region of the MALDI matrix, i.e. typically below m/z 1000. Figure S4. Enlargements of negative mode MALDI-ISD FT-ICR mass spectra of the glycoconjugates EcoO2, EcoO6A, and EcoO25B. The c'7 fragment ion, generated from the cleavage of the peptide bond between Gln7 and the potentially glycosylated Asn8, was detected in all mass spectra. Figure S5. Enlargements of negative mode MALDI-ISD FT-ICR mass spectra of the glycoconjugates EcoO2, EcoO6A, and EcoO25B. The c'9 fragment ion included the potentially glycosylated Asn8. This fragment ion, as well as larger c'-type ions, was not detected in the mass spectrum of EcoO6A indicating a high glycosylation site occupancy at Asn8.  Figure S7. Enlargement of the MALDI-ISD FT-ICR mass spectra of the glycoconjugates EcoO2, EcoO6A, and EcoO25B in the m/z-range 3000-8000. Sodiated B ions corresponding to up to 9 repeating units were detected in this m/z-range. The mass spectrum of EcoO25B showed a higher complexity due to the presence of fragment ions generated from the partial loss of acetylation and C-type fragmentation. Not annotated peaks are fragment ions generated from the cleavage of one or more different glycosidic bonds (See Figures 4 and S9-S10).  Figure S9. Enlargement of the MALDI-ISD FT-ICR mass spectrum of the glycoconjugate EcoO6A (see Figure 3) in the m/z-range between sodiated B-ions corresponding to 1RU and 2RU. The cleavage of one or more glycosidic bonds led to the formation of the fragment ions highlighted in red. Different isomeric fragment ions may exist for each assigned peak. Figure S10. Enlargement of the MALDI-ISD FT-ICR mass spectrum of the glycoconjugate EcoO25B (see Figure 3) in the m/z-range between sodiated B-ions corresponding to 1RU and 2RU. The cleavage of one or more glycosidic bonds led to the formation of the fragment ions highlighted in red. Different isomeric fragment ions may exist for each assigned peak. * indicates fragment ions that were generated from repeat unit variants lacking one (or more) acetyl groups.