Identifying Carbohydrate Ligands of a Norovirus P Particle using a Catch and Release Electrospray Ionization Mass Spectrometry Assay

  • Ling Han
  • Elena N. Kitova
  • Ming Tan
  • Xi Jiang
  • John S. Klassen
Research Article


Noroviruses (NoVs), the major cause of epidemic acute gastroenteritis, recognize human histo-blood group antigens (HBGAs), which are present as free oligosaccharides in bodily fluid or glycolipids and glycoproteins on the surfaces of cells. The subviral P particle formed by the protruding (P) domain of the NoV capsid protein serves as a useful model for the study NoV–HBGA interactions. Here, we demonstrate the application of a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against the P particle to rapidly identify NoV ligands and potential inhibitors. Carbohydrate libraries of 50 and 146 compounds, which included 18 and 24 analogs of HBGA receptors, respectively, were screened against the P particle of VA387, a member of the predominant GII.4 NoVs. Deprotonated ions corresponding to the P particle bound to carbohydrates were isolated and subjected to collision-induced dissociation to release the ligands in their deprotonated forms. The released ligands were identified by ion mobility separation followed by mass analysis. All 13 and 16 HBGA ligands with intrinsic affinities >500 M–1 were identified in the 50 and the 146 compound libraries, respectively. Furthermore, screening revealed interactions with a series of oligosaccharides with structures found in the cell wall of mycobacteria and human milk. The affinities of these newly discovered ligands are comparable to those of the HBGA receptors, as estimated from the relative abundance of released ligand ions.

Key words

Catch and release Electrospray ionization mass spectrometry Norovirus P particle Carbohydrate 



The authors thank the Alberta Glycomics Centre and the National Institutes of Health of the Unites States of America for supporting this research. They thank Professors T. Lowary (University of Alberta) and C.-C. Ling (University of Calgary), as well as Alberta Innovates Technology Futures for generously providing some of the carbohydrates used in this work. L.H. also acknowledges an Alberta Innovates Graduate Student Scholarship.

Supplementary material

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ESM 1 (PDF 402 kb)


  1. 1.
    Patel, M.M., Widdowson, M.-A., Glass, R.I., Akazawa, K., Vinje, J., Parashar, U.D.: Systematic literature review of role of noroviruses in sporadic gastroenteritis. Emerg. Infect. Dis 14, 1224–1231 (2008)CrossRefGoogle Scholar
  2. 2.
    Hutson, A.M., Atmar, R.L., Marcus, D.M., Estes, M.K.: Norwalk virus-like particle hemagglutination by binding to H histo-blood group antigens. J. Virol 77, 405–415 (2003)CrossRefGoogle Scholar
  3. 3.
    Huang, P., Farkas, T., Marionneau, S., Zhong, W., Ruvoen-Clouet, N., Morrow, A.L., Altaye, M., Pickering, L.K., Newburg, D.S., LePendu, J., Jiang, X.: Noroviruses bind to human ABO, Lewis, and secretor histo-blood group antigens: Identification of 4 distinct strain-specific patterns. J. Infect. Dis 188, 19–31 (2003)CrossRefGoogle Scholar
  4. 4.
    Huang, P., Farkas, T., Zhong, W., Thornton, S., Morrow, A.L., Jiang, X.: Norovirus and histo-blood group antigens: demonstration of a wide spectrum of strain specificities and classification of two major binding groups among multiple binding patterns. J. Virol 79, 6714–6722 (2005)CrossRefGoogle Scholar
  5. 5.
    Tan, M., Jiang, X.: Norovirus and its histo-blood group antigen receptors: an answer to a historical puzzle. Trends Microbiol 13, 285–293 (2005)CrossRefGoogle Scholar
  6. 6.
    Fiege, B., Rademacher, C., Cartmell, J., Kitov, P.I., Parra, F., Peters, T.: Molecular details of the recognition of blood group antigens by a human norovirus as determined by STD NMR spectroscopy. Angew. Chem. Int. Ed 51, 928–932 (2012)CrossRefGoogle Scholar
  7. 7.
    Tan, M., Jiang, X.: Norovirus-host interaction: implications for disease control and prevention. Expert Rev. Mol. Med 9, 1–22 (2007)CrossRefGoogle Scholar
  8. 8.
    Tan, M., Jiang, X.: Norovirus gastroenteritis, carbohydrate receptors, and animal models. PLoS Pathogens 6, e1000983 (2010)CrossRefGoogle Scholar
  9. 9.
    Tan, M., Jiang, X.: Norovirus-host interaction: multi-selections by human histo-blood group antigens. Trends Microbiol 19, 382–388 (2011)CrossRefGoogle Scholar
  10. 10.
    Oriol, R.: Genetic-control of the fucosylation of ABH precursor chains—evidence for new epistatic interactions in different cells and tissues. J. Immunogenet 17, 235–245 (1990)CrossRefGoogle Scholar
  11. 11.
    Ravn, V., Dabelsteen, E.: Tissue distribution of histo-blood group antigens. APMIS 108, 1–28 (2000)CrossRefGoogle Scholar
  12. 12.
    Han, L., Kitov, P.I., Kitova, E.N., Tan, M., Wang, L., Xia, M., Jiang, X., Klassen, J.S.: Affinities of recombinant norovirus P dimers for human blood group antigens. Glycobiology 23, 276–285 (2013)CrossRefGoogle Scholar
  13. 13.
    Prasad, B.V.V., Hardy, M.E., Dokland, T., Bella, J., Rossmann, M.G., Estes, M.K.: X-ray crystallographic structure of the Norwalk virus capsid. Science 286, 287–290 (1999)CrossRefGoogle Scholar
  14. 14.
    Tan, M., Hegde, R.S., Jiang, X.: The P domain of norovirus capsid protein forms dimer and binds to histo-blood group antigen receptors. J. Virol 78, 6233–6242 (2004)CrossRefGoogle Scholar
  15. 15.
    Tan, M., Huang, P., Meller, J., Zhong, W., Farkas, T., Jiang, X.: Mutations within the P2 domain of norovirus capsid affect binding to human histo-blood group antigens: Evidence for a binding pocket. J. Virol 77, 12562–12571 (2003)CrossRefGoogle Scholar
  16. 16.
    Jiang, X., Wang, M., Graham, D.Y., Estes, M.K.: Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein. J. Virol 66, 6527–6532 (1992)Google Scholar
  17. 17.
    Shoemaker, G.K., van Duijn, E., Crawford, S.E., Uetrecht, C., Baclayon, M., Roos, W.H., Wuite, G.J.L., Estes, M.K., Prasad, B.V.V., Heck, A.J.R.: Norwalk virus assembly and stability monitored by mass spectrometry. Mol. Cell. Proteom 9, 1742–1751 (2010)CrossRefGoogle Scholar
  18. 18.
    Tan, M., Meller, J., Jiang, X.: C-terminal arginine cluster is essential for receptor binding of norovirus capsid protein. J. Virol 80, 7322–7331 (2006)CrossRefGoogle Scholar
  19. 19.
    Tan, M., Xia, M., Cao, S., Huang, P., Farkas, T., Meller, J., Hegde, R.S., Li, X., Rao, Z., Jiang, X.: Elucidation of strain-specific interaction of a GII-4 norovirus with HBGA receptors by site-directed mutagenesis study. Virology 379, 324–334 (2008)CrossRefGoogle Scholar
  20. 20.
    Bu, W., Mamedova, A., Tan, M., Xia, M., Jiang, X., Hegde, R.S.: Structural basis for the receptor binding specificity of Norwalk virus. J. Virol. 82, 5340–5347 (2008) native mass spectrometry. J. Struct. Biol. 177, 273–282 (2012)Google Scholar
  21. 21.
    Cao, S., Lou, Z., Tan, M., Chen, Y., Liu, Y., Zhang, Z., Zhang, X.C., Jiang, X., Li, X., Rao, Z.: Structural basis for the recognition of blood group trisaccharides by norovirus. J. Virol 81, 5949–5957 (2007)CrossRefGoogle Scholar
  22. 22.
    Choi, J.M., Hutson, A.M., Estes, M.K., Prasad, B.V.V.: Atomic resolution structural characterization of recognition of histo-blood group antigens by Norwalk virus. Proc. Natl. Acad. Sci. U.S.A 105, 9175–9180 (2008)CrossRefGoogle Scholar
  23. 23.
    Chen, Y., Tan, M., Xia, M., Hao, N., Zhang, X.C., Huang, P., Jiang, X., Li, X., Rao, Z.: Crystallography of a Lewis-binding norovirus, elucidation of strain-specificity to the Polymorphic Human Histo-Blood Group Antigens. PLoS Pathog 7, e1002152 (2011)CrossRefGoogle Scholar
  24. 24.
    Hansman, G.S., Biertumpfel, C., Georgiev, I., McLellan, J.S., Chen, L., Zhou, T.Q., Katayama, K., Kwong, P.D.: Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability. J. Virol 85, 6687–6701 (2011)CrossRefGoogle Scholar
  25. 25.
    Shanker, S., Choi, J.M., Sankaran, B., Atmar, R.L., Estes, M.K., Prasad, B.V.V.: Structural analysis of histo-blood group antigen binding specificity in a norovirus GII.4 epidemic variant: implications for epochal evolution. J. Virol 85, 8635–8645 (2011)CrossRefGoogle Scholar
  26. 26.
    Kubota, T., Kumagai, A., Ito, H., Furukawa, S., Someya, Y., Takeda, N., Ishii, K., Wakita, T., Narimatsu, H., Shirato, H.: Structural basis for the recognition of Lewis antigens by genogroup I norovirus. J. Virol 86, 11138–11150 (2012)CrossRefGoogle Scholar
  27. 27.
    Tan, M., Jiang, X.: The P domain of norovirus capsid protein forms a subviral particle that binds to histo-blood group antigen receptors. J. Virol 79, 14017–14030 (2005)CrossRefGoogle Scholar
  28. 28.
    Tan, M., Fang, P., Chachiyo, T., Xia, M., Huang, P., Fang, Z., Jiang, W., Jiang, X.: Noroviral P particle: structure, function and applications in virus–host interaction. Virology 382, 115–123 (2008)CrossRefGoogle Scholar
  29. 29.
    Tan, M., Huang, P., Xia, M., Fang, P.A., Zhong, W., McNeal, M., Wei, C., Jiang, W., Jiang, X.: Norovirus P particle, a novel platform for vaccine development and antibody production. J. Virol 85, 753–764 (2011)CrossRefGoogle Scholar
  30. 30.
    Tan, M., Fang, P.A., Xia, M., Chachiyo, T., Jiang, W., Jiang, X.: Terminal modifications of norovirus P domain resulted in a new type of subviral particles, the small P particles. Virology 410, 345–352 (2011)CrossRefGoogle Scholar
  31. 31.
    Tan, M., Zhong, W., Song, D., Thornton, S., Jiang, X.: E. coli-expressed recombinant norovirus capsid proteins maintain authentic antigenicity and receptor binding capability. J. Med. Virol 74, 641–649 (2004)CrossRefGoogle Scholar
  32. 32.
    Tan, M., Xia, M., Chen, Y., Bu, W., Hegde, R.S., Meller, J., Li, X., Jiang, X.: Conservation of carbohydrate binding interfaces: evidence of human HBGA selection in norovirus evolution. PLoS One 4, e5058 (2009)CrossRefGoogle Scholar
  33. 33.
    Shang, J., Cheng, F., Dubey, M., Kaplan, J.M., Rawal, M., Jiang, X., Newburg, D.S., Sullivan, P.A., Andrade, R.B., Ratner, D.M.: An organophosphonate strategy for functionalizing silicon photonic biosensors. Langmuir 28, 3338–3344 (2012)CrossRefGoogle Scholar
  34. 34.
    Zhang, X., Dai, Y., Zhong, W., Tan, M., Lv, Z., Zhou, Y., Jiang, X.: Tannic acid inhibited norovirus binding to HBGA receptors, a study of 50 Chinese medicinal herbs. Bioorg. Med. Chem 20, 1616–1623 (2012)CrossRefGoogle Scholar
  35. 35.
    Tan, M., Jiang, X.: Norovirus P particle, a subviral nanoparticle for vaccine development against norovirus, rotavirus and influenza virus. Nanomedicine 7, 1–9 (2012)CrossRefGoogle Scholar
  36. 36.
    Xia, M., Tan, M., Wei, C., Zhong, W., Wang, L., McNeal, M., Jiang, X.: A candidate dual vaccine against influenza and noroviruses. Vaccine 29, 7670–7677 (2011)CrossRefGoogle Scholar
  37. 37.
    Fang, H., Tan, M., Xia, M., Wang, L., Jiang, X.: Norovirus P particle efficiently elicits innate, humoral, and cellular immunity. PLoS One 8, e63269 (2013)CrossRefGoogle Scholar
  38. 38.
    Dai, Y., Wang, Y., Zhang, X., Tan, M., Xia, M., Wu, X., Jiang, X., Nie, J.: Evaluation of anti-norovirus IgY from egg yolk of chickens immunized with norovirus P particles. J. Virol. Methods 186, 126–131 (2012)CrossRefGoogle Scholar
  39. 39.
    Dai, Y., Zhang, X., Tan, M., Huang, P., Lei, W., Fang, H., Zhong, W., Jiang, X.: A dual chicken IgY against rotavirus and norovirus. Antivir. Res 97, 293–300 (2013)CrossRefGoogle Scholar
  40. 40.
    Wang, L., Huang, P., Fang, H., Xia, M., Zhong, W., McNeal, M.M., Jiang, X., Tan, M.: Polyvalent complexes for vaccine development. Biomaterials 34, 4480–4492 (2013)CrossRefGoogle Scholar
  41. 41.
    Feng, X., Jiang, X.: Library screen for inhibitors targeting norovirus binding to histo-blood group antigen receptors. Antimicrob. Agents Chemother 51, 324–331 (2007)CrossRefGoogle Scholar
  42. 42.
    Rademacher, C., Guiard, J., Kitov, P.I., Fiege, B., Dalton, K.P., Parra, F., Bundle, D.R., Peters, T.: Targeting norovirus infection-multivalent entry inhibitor design based on NMR experiments. Chem. Eur. J 17, 7442–7453 (2011)CrossRefGoogle Scholar
  43. 43.
    Zhang, X., Tan, M., Chhabra, M., Dai, Y., Meller, J., Jiang, X.: Inhibition of histo-blood group antigen binding as a novel strategy to block norovirus infections. PLoS One 8, e69379 (2013)CrossRefGoogle Scholar
  44. 44.
    Cederkvist, F., Zamfir, A.D., Bahrke, S., Eijsink, V.G.H., Sorlie, M., Peter-Katalinic, J., Peter, M.G.: Identification of a high-affinity-binding oligosaccharide by (+) nanoelectrospray quadrupole time-of-flight tandem mass spectrometry of a noncovalent enzyme-ligand complex. Angew. Chem. Int. Ed 45, 2429–2434 (2006)CrossRefGoogle Scholar
  45. 45.
    Abzalimov, R.R., Dubin, P.L., Kaltashov, I.A.: Glycosaminoglycans as naturally occurring combinatorial libraries: spectrometry-based strategy for characterization of anti-thrombin interaction strategy with low molecular weight heparin and heparin oligomers. Anal. Chem 79, 6055–6063 (2007)CrossRefGoogle Scholar
  46. 46.
    El-Hawiet, A., Shoemaker, G.K., Daneshfar, R., Kitova, E.N., Klassen, J.S.: Applications of a catch and release electrospray ionization mass spectrometry assay for carbohydrate library screening. Anal. Chem 84, 50–58 (2012)CrossRefGoogle Scholar
  47. 47.
    Bereszczak, J.Z., Barbu, I.M., Tan, M., Xia, M., Jiang, X., van Duijn, E., Heck, A.J.R.: Structure, stability, and dynamics of norovirus P domain derived protein complexes studied by native mass spectrometry. J. Struct. Biol 177, 273–282 (2012)CrossRefGoogle Scholar
  48. 48.
    Sun, J.X., Kitova, E.N., Wang, W.J., Klassen, J.S.: Method for distinguishing specific from nonspecific protein–ligand complexes in nanoelectrospray ionization mass spectrometry. Anal. Chem 78, 3010–3018 (2006)CrossRefGoogle Scholar
  49. 49.
    Kitova, E.N., El-Hawiet, A., Schnier, P.D., Klassen, J.S.: Reliable determinations of protein-ligand interactions by direct ESI-MS measurements. Are we there yet? J. Am. Soc. Mass Spectrom 23, 431–441 (2012)CrossRefGoogle Scholar
  50. 50.
    Hansman, G.S., Shahzad-ul-Hussan, S., McLellan, J.S., Chuang, G.-Y., Georgiev, I., Shimoike, T., Katayama, K., Bewley, C.A., Kwong, P.D.: Structural basis for norovirus inhibition and fucose mimicry by citrate. J. Virol 86, 284–292 (2012)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2013

Authors and Affiliations

  • Ling Han
    • 1
  • Elena N. Kitova
    • 1
  • Ming Tan
    • 2
    • 3
  • Xi Jiang
    • 2
    • 3
  • John S. Klassen
    • 1
  1. 1.Alberta Glycomics Centre and Department of ChemistryUniversity of AlbertaEdmontonCanada
  2. 2.Division of Infectious DiseasesCincinnati Children’s Hospital Medical CenterCincinnatiUSA
  3. 3.Department of PediatricsUniversity of Cincinnati College of MedicineCincinnatiUSA

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