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Mass photometry: A powerful tool for carbohydrates-proteins conjugation monitoring and glycoconjugates molecular mass determination

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Abstract

Glycoconjugate vaccines are important additions to the existing means for prevention of diseases caused by bacterial and viral pathogens. Conjugating carbohydrates to proteins is a crucial step in the development of these vaccines. Traditional mass spectrometry techniques, such as MALDI-TOF and SELDI-TOF, have difficulties in detecting glycoconjugates with high molecular masses. Mass photometry (MP) is a single-molecule technique that has been recently developed, which allows mass measurements of individual molecules and generates mass distributions based on hundreds to thousands of these measurements. In this study, we evaluated the performance of MP in monitoring carbohydrate-protein conjugation reactions and characterization of conjugates. Three different glycoconjugates were prepared from carrier protein BSA, and one from a large protein complex, a virus capsid with 3.74 MDa molecular mass. The masses measured by MP were consistent with those obtained by SELDI-TOF-MS and SEC-MALS. The conjugation of BSA dimer to carbohydrate antigen was also successfully characterized. This study shows that the MP technique is a promising alternative to methods developed earlier for monitoring glycoconjugation reactions and characterization of glycoconjugates. It measures intact molecules in solution and it is highly accurate over a wide mass range. MP requires only a very small amount of sample and has no specific buffer constraints. Other MP advantages include minimal cost of consumables and rapid data collection and analysis. Its advantages over other methods make it a valuable tool for researchers in the glycoconjugation field.

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Availability of data and materials

The data set generated during the current study are available from the corresponding author upon reasonable request.

Reference

  1. Dick, W.E., Jr., Beurret, M.: Glycoconjugates of bacterial carbohydrate antigens. A survey and consideration of design and preparation factors. Contrib. Microbiol. Immunol. 10, 48–114 (1989)

    PubMed  Google Scholar 

  2. Robbins, J.B., et al.: Synthesis, characterization, and immunogenicity in mice of Shigella sonnei O-specific oligosaccharide-core-protein conjugates. Proc. Natl. Acad. Sci. USA 106(19), 7974–7978 (2009). https://doi.org/10.1073/pnas.0900891106

    Article  PubMed  PubMed Central  Google Scholar 

  3. Harvey, D.J.: Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2019–2020. Mass Spectrom. Rev. (2022). https://doi.org/10.1002/mas.21806

  4. Wright, G.L., Jr., et al.: Proteinchip(R) surface enhanced laser desorption/ionization (SELDI) mass spectrometry: a novel protein biochip technology for detection of prostate cancer biomarkers in complex protein mixtures. Prostate Cancer Prostatic. Dis. 2(5–6), 264–276 (1999). https://doi.org/10.1038/sj.pcan.4500384

    Article  CAS  PubMed  Google Scholar 

  5. Kamath, V.P., Diedrich, P., Hindsgaul, O.: Use of diethyl squarate for the coupling of oligosaccharide amines to carrier proteins and characterization of the resulting neoglycoproteins by MALDI-TOF mass spectrometry. Glycoconj. J. 13(2), 315–319 (1996). https://doi.org/10.1007/BF00731506

    Article  CAS  PubMed  Google Scholar 

  6. Ma, X., et al.: Neoglycoconjugates from synthetic tetra- and hexasaccharides that mimic the terminus of the O-PS of Vibrio cholerae O:1, serotype Inaba. Org. Biomol. Chem. 1(5), 775–784 (2003). https://doi.org/10.1039/b211660

    Article  CAS  PubMed  Google Scholar 

  7. Leney, A.C., Heck, A.J.: Native Mass Spectrometry: What is in the Name? J. Am. Soc. Mass Spectrom. 28(1), 5-13 (2017). https://doi.org/10.1007/s13361-016-1545-3

  8. Mehmood, S., Allison, T.M., Robinson, C.V.: Mass spectrometry of protein complexes: from origins to applications. Annu. Rev. Phys. Chem. 66. 453-474. https://doi.org/10.1146/annurev-physchem-040214-121732

  9. Tamara, S., den Boer, M.A., Heck, A.J.R.: High-Resolution Native Mass Spectrometry. Chem. Rev. 122(8), 7269-7326 (2022). https://doi.org/10.1021/acs.chemrev.1c00212

  10. Chernyak, A., et al.: Conjugating oligosaccharides to proteins by squaric acid diester chemistry: rapid monitoring of the progress of conjugation, and recovery of the unused ligand. Carbohydr. Res. 330(4), 479–486 (2001).

  11. Xu, P., et al.: Conjugate Vaccines from Bacterial Antigens by Squaric Acid Chemistry: A Closer Look. Chembiochemistry 18(8), 799–815 (2017). https://doi.org/10.1002/cbic.201600699

  12. Pfister, H.B., et al.: Conjugation of Synthetic Oligosaccharides to Proteins by Squaric Acid Chemistry. Methods Mol. Biol. 1954, 77-88 (2019). https://doi.org/10.1007/978-1-4939-9154-9_7

  13. Xu, P., Kovac, P.: Direct Conjugation of Bacterial Polysaccharides to Proteins by Squaric Acid. Chem. Methods Mol. Biol. 1954, 89-98 (2019). https://doi.org/10.1007/978-1-4939-9154-9_8

  14. Cole, D., et al.: Label-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering Microscopy. ACS Photonics 4(2), 211-216 (2017). https://doi.org/10.1021/acsphotonics.6b00912

  15. Young, G., et al.: Quantitative mass imaging of single biological macromolecules. Science 360(6387), 423–427 (2018). https://doi.org/10.1126/science.aar5839

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nicolai, T., et al.: Molar mass characterization of DNA fragments by gel permeation chromatography using a low-angle laser light-scattering detector. J. Chromatogr. 389(1), 286–292 (1987). https://doi.org/10.1016/s0021-9673(01)94436-x

    Article  CAS  PubMed  Google Scholar 

  17. Theisen, A.J., Deacon, C., Harding, M.P.: S.E., Refractive Increment Data-Book for Polymer and Biomolecular Scientists. Nottingham University Press (2000)

  18. Zhao, H., Brown, P.H., Schuck, P.: On the distribution of protein refractive index increments Biophys. J. 100(9), 2309–2317 (2011). https://doi.org/10.1016/j.bpj.2011.03.004

  19. Wu, D., Piszczek, G.: Measuring the affinity of protein-protein interactions on a single-molecule level by mass photometry. Anal. Biochem. 592, 113575 (2020). https://doi.org/10.1016/j.ab.2020.113575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Li, Y., Struwe, W.B., Kukura, P.: Single molecule mass photometry of nucleic acids. Nucleic Acids Res. 48(17), e97 (2020). https://doi.org/10.1093/nar/gkaa632

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Olerinyova, A., et al.: Mass Photometry of Membrane Proteins.  Chemistry 7(1), 224–236 (2021). https://doi.org/10.1016/j.chempr.2020.11.011

    Article  CAS  Google Scholar 

  22. Wu, D., et al.: Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry. Gene Ther. (2022). https://doi.org/10.1038/s41434-021-00311-4

  23. Wu, D., Piszczek, G.: Standard protocol for mass photometry experiments. Eur. Biophys. J. 50(3–4), 403–409 (2021). https://doi.org/10.1007/s00249-021-01513-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hou, S.J., Saksena, R., Kovac, P.: Preparation of glycoconjugates by dialkyl squarate chemistry revisited. Carbohydr. Res. 343(2), 196–210 (2008). https://doi.org/10.1016/j.carres.2007.10.015

    Article  CAS  PubMed  Google Scholar 

  25. Xu, P., et al.: Simple, direct conjugation of bacterial O-SP-core antigens to proteins: development of cholera conjugate vaccines. Bioconjug. Chem. 22(10), 2179–2185 (2011). https://doi.org/10.1021/bc2001984

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bongat, A.F., et al.: Multimeric bivalent immunogens from recombinant tetanus toxin HC fragment, synthetic hexasaccharides, and a glycopeptide adjuvant. Glycoconj. J. 27(1), 69–77 (2010). https://doi.org/10.1007/s10719-009-9259-4

    Article  CAS  PubMed  Google Scholar 

  27. Kendrick, B.S., et al.: Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states. Anal. Biochem. 299(2), 136–146 (2001). https://doi.org/10.1006/abio.2001.5411

    Article  CAS  PubMed  Google Scholar 

  28. Barer, R., Joseph, S.: Refractometry of Living Cells. Quart. J. Microcop. Sci. 95, 399–423 (1954)

    CAS  Google Scholar 

  29. Tumolo, T., Angnes, L., Baptista, M.S.: Determination of the refractive index increment (dn/dc) of molecule and macromolecule solutions by surface plasmon resonance. Anal. Biochem. 333(2), 273-279 (2004). https://doi.org/10.1016/j.ab.2004.06.010

  30. Nobbmann, U.: Refractive index increment dn/dc values. Available from (2013). https://www.materials-talks.com/refractive-index-increment-dndc-values/

  31. Rashidijahanabad, Z., et al.: Virus-like Particle Display of Vibrio cholerae O-Specific Polysaccharide as a Potential Vaccine against Cholera ACS. Infect. Dis. 8(3), 574–583 (2022). https://doi.org/10.1021/acsinfecdis.1c00585

    Article  CAS  Google Scholar 

  32. Sommer, J.M., et al.: Quantification of adeno-associated virus particles and empty capsids by optical density measurement. Mol. Ther. 7(1), 122–128 (2003).

  33. Beirne, J., Truchan, H., Rao, L.: Development and qualification of a size exclusion chromatography coupled with multiangle light scattering method for molecular weight determination of unfractionated heparin. Anal. Bioanal. Chem. 399(2), 717–725 (2011). https://doi.org/10.1007/s00216-010-4187-5

    Article  CAS  PubMed  Google Scholar 

  34. Folta-Stogniew, E., Williams, K.R.: Determination of molecular masses of proteins in solution: Implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory. J. Biomol. Tech. 10(2), 51–63 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Porterfield, J.Z., Zlotnick, A.: A simple and general method for determining the protein and nucleic acid content of viruses by UV absorbance. Virology 407(2), 281-288 (2010). https://doi.org/10.1016/j.virol.2010.08.015

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Acknowledgment

We thank Dr. Duck-Yeon Lee in the Biochemistry Core of NHLBI for discussion.

Funding

This work was supported by the intramural program of the NHLBI and NIDDK, NIH, and extramural program of NIAID, NIH (R37106878).

Author information

Authors and Affiliations

  1. Biophysics Core Facility, NHLBI, NIH, Bethesda, MD, 20892, USA

    Di Wu & Grzegorz Piszczek

  2. Laboratory of Bioorganic Chemistry, NIDDK, NIH, Bethesda, MD, 20892, USA

    Peng Xu & Pavol Kováč

  3. Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA

    Meagan Kelly & Edward T. Ryan

  4. Department of Medicine, Harvard Medical School, Boston, MA, USA

    Edward T. Ryan

  5. Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA

    Edward T. Ryan

Authors
  1. Di Wu
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  2. Peng Xu
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  3. Meagan Kelly
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  4. Edward T. Ryan
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  5. Pavol Kováč
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  6. Grzegorz Piszczek
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Contributions

D.W., P.X. and G.P. conceived the study; P.X., M.K. and E.R. performed the conjugation experiments. D.W. performed the MP experiments. D.W., P.X., P.K. and G.P. wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to Di Wu.

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Wu, D., Xu, P., Kelly, M. et al. Mass photometry: A powerful tool for carbohydrates-proteins conjugation monitoring and glycoconjugates molecular mass determination. Glycoconj J 40, 401–412 (2023). https://doi.org/10.1007/s10719-023-10126-7

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  • DOI: https://doi.org/10.1007/s10719-023-10126-7

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