Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2001 Dec;81(6):3503–3509. doi: 10.1016/S0006-3495(01)75981-4

Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach.

M A Tito 1, J Miller 1, N Walker 1, K F Griffin 1, E D Williamson 1, D Despeyroux-Hill 1, R W Titball 1, C V Robinson 1
PMCID: PMC1301805  PMID: 11721011

Abstract

Using a combination of nanoflow-electrospray ionization and time-of-flight mass spectrometry we have analyzed the oligomeric state of the recombinant V antigen from Yersinia pestis, the causative agent of plague. The mass spectrometry results show that at pH 6.8 the V antigen in solution exists predominantly as a dimer and a weakly associated tetramer. A monoclonal antibody 7.3, raised against the V antigen, gave rise to mass spectra containing a series of well-resolved charge states at m/z 6000. After addition of aliquots of solution containing V antigen in substoichiometric and molar equivalents, the spectra revealed that two molecules of the V antigen bind to the antibody. Collision-induced dissociation of the antibody-antigen complex results in the selective release of the dimer from the complex supporting the proposed 1:2 antibody:antigen stoichiometry. Control experiments with the recombinant F1 antigen, also from Yersinia pestis, establish that the antibody is specific for the V antigen because no complex with F1 was detected even in the presence of a 10-fold molar excess of F1 antigen. More generally this work demonstrates a rapid means of assessing antigen subunit interactions as well as the stoichiometry and specificity of binding in antibody-antigen complexes.

Full Text

The Full Text of this article is available as a PDF (139.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bennett K. L., Smith S. V., Lambrecht R. M., Truscott R. J., Sheil M. M. Rapid characterization of chemically-modified proteins by electrospray mass spectrometry. Bioconjug Chem. 1996 Jan-Feb;7(1):16–22. doi: 10.1021/bc950064c. [DOI] [PubMed] [Google Scholar]
  2. Biemann K. Sequencing of peptides by tandem mass spectrometry and high-energy collision-induced dissociation. Methods Enzymol. 1990;193:455–479. doi: 10.1016/0076-6879(90)93433-l. [DOI] [PubMed] [Google Scholar]
  3. Downard K. M. Contributions of mass spectrometry to structural immunology. J Mass Spectrom. 2000 Apr;35(4):493–503. doi: 10.1002/(SICI)1096-9888(200004)35:4<493::AID-JMS987>3.0.CO;2-I. [DOI] [PubMed] [Google Scholar]
  4. Eyles J. E., Williamson E. D., Spiers I. D., Stagg A. J., Jones S. M., Alpar H. O. Generation of protective immune responses to plague by mucosal administration of microsphere coencapsulated recombinant subunits. J Control Release. 2000 Jan 3;63(1-2):191–200. doi: 10.1016/s0168-3659(99)00197-2. [DOI] [PubMed] [Google Scholar]
  5. Fändrich M., Tito M. A., Leroux M. R., Rostom A. A., Hartl F. U., Dobson C. M., Robinson C. V. Observation of the noncovalent assembly and disassembly pathways of the chaperone complex MtGimC by mass spectrometry. Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14151–14155. doi: 10.1073/pnas.240326597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hill J., Leary S. E., Griffin K. F., Williamson E. D., Titball R. W. Regions of Yersinia pestis V antigen that contribute to protection against plague identified by passive and active immunization. Infect Immun. 1997 Nov;65(11):4476–4482. doi: 10.1128/iai.65.11.4476-4482.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kebarle P. A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry. J Mass Spectrom. 2000 Jul;35(7):804–817. doi: 10.1002/1096-9888(200007)35:7<804::AID-JMS22>3.0.CO;2-Q. [DOI] [PubMed] [Google Scholar]
  8. Krebs J. F., Siuzdak G., Dyson H. J., Stewart J. D., Benkovic S. J. Detection of a catalytic antibody species acylated at the active site by electrospray mass spectrometry. Biochemistry. 1995 Jan 24;34(3):720–723. doi: 10.1021/bi00003a002. [DOI] [PubMed] [Google Scholar]
  9. Leary S. E., Williamson E. D., Griffin K. F., Russell P., Eley S. M., Titball R. W. Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague. Infect Immun. 1995 Aug;63(8):2854–2858. doi: 10.1128/iai.63.8.2854-2858.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Nettleton E. J., Sunde M., Lai Z., Kelly J. W., Dobson C. M., Robinson C. V. Protein subunit interactions and structural integrity of amyloidogenic transthyretins: evidence from electrospray mass spectrometry. J Mol Biol. 1998 Aug 21;281(3):553–564. doi: 10.1006/jmbi.1998.1937. [DOI] [PubMed] [Google Scholar]
  11. Robinson C. V., Gross M., Eyles S. J., Ewbank J. J., Mayhew M., Hartl F. U., Dobson C. M., Radford S. E. Conformation of GroEL-bound alpha-lactalbumin probed by mass spectrometry. Nature. 1994 Dec 15;372(6507):646–651. doi: 10.1038/372646a0. [DOI] [PubMed] [Google Scholar]
  12. Rostom A. A., Fucini P., Benjamin D. R., Juenemann R., Nierhaus K. H., Hartl F. U., Dobson C. M., Robinson C. V. Detection and selective dissociation of intact ribosomes in a mass spectrometer. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5185–5190. doi: 10.1073/pnas.97.10.5185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Rostom A. A., Robinson C. V. Disassembly of intact multiprotein complexes in the gas phase. Curr Opin Struct Biol. 1999 Feb;9(1):135–141. doi: 10.1016/s0959-440x(99)80018-9. [DOI] [PubMed] [Google Scholar]
  14. Verentchikov A. N., Ens W., Standing K. G. Reflecting time-of-flight mass spectrometer with an electrospray ion source and orthogonal extraction. Anal Chem. 1994 Jan 1;66(1):126–133. doi: 10.1021/ac00073a022. [DOI] [PubMed] [Google Scholar]
  15. Williamson E. D., Eley S. M., Stagg A. J., Green M., Russell P., Titball R. W. A sub-unit vaccine elicits IgG in serum, spleen cell cultures and bronchial washings and protects immunized animals against pneumonic plague. Vaccine. 1997 Jul;15(10):1079–1084. doi: 10.1016/s0264-410x(96)00303-9. [DOI] [PubMed] [Google Scholar]
  16. van Berkel W. J., van den Heuvel R. H., Versluis C., Heck A. J. Detection of intact megaDalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry. Protein Sci. 2000 Mar;9(3):435–439. doi: 10.1110/ps.9.3.435. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES