Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach.
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 pred...
| Main Authors: | , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
2001
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| _version_ | 1797085059680829440 |
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| author | Tito, M Miller, J Walker, N Griffin, K Williamson, E Despeyroux-Hill, D Titball, R Robinson, C |
| author_facet | Tito, M Miller, J Walker, N Griffin, K Williamson, E Despeyroux-Hill, D Titball, R Robinson, C |
| author_sort | Tito, M |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T02:03:49Z |
| format | Journal article |
| id | oxford-uuid:9e489ede-9aaf-4796-b5a1-e49a33dfe88e |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T02:03:49Z |
| publishDate | 2001 |
| record_format | dspace |
| spelling | oxford-uuid:9e489ede-9aaf-4796-b5a1-e49a33dfe88e2022-03-27T00:49:03ZProbing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:9e489ede-9aaf-4796-b5a1-e49a33dfe88eEnglishSymplectic Elements at Oxford2001Tito, MMiller, JWalker, NGriffin, KWilliamson, EDespeyroux-Hill, DTitball, RRobinson, CUsing 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. |
| spellingShingle | Tito, M Miller, J Walker, N Griffin, K Williamson, E Despeyroux-Hill, D Titball, R Robinson, C Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach. |
| title | Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach. |
| title_full | Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach. |
| title_fullStr | Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach. |
| title_full_unstemmed | Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach. |
| title_short | Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach. |
| title_sort | probing molecular interactions in intact antibody antigen complexes an electrospray time of flight mass spectrometry approach |
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