ac0c02237_si_001.pdf (4.54 MB)
Download file

Mapping a Conformational Epitope of Hemagglutinin A Using Native Mass Spectrometry and Ultraviolet Photodissociation

Download (4.54 MB)
journal contribution
posted on 12.08.2020, 14:38 by M. Rachel Mehaffey, Jiwon Lee, Jiwon Jung, Michael B. Lanzillotti, Edwin E. Escobar, Keith R. Morgenstern, George Georgiou, Jennifer S. Brodbelt
As the importance of effective vaccines and the role of protein therapeutics in the drug industry continue to expand, alternative strategies to characterize protein complexes are needed. Mass spectrometry (MS) in conjunction with enzymatic digestion or chemical probes has been widely used for mapping binding epitopes at the molecular level. However, advances in instrumentation and application of activation methods capable of accessing higher energy dissociation pathways have recently allowed direct analysis of protein complexes. Here we demonstrate a workflow utilizing native MS and ultraviolet photodissociation (UVPD) to map the antigenic determinants of a model antibody–antigen complex involving hemagglutinin (HA), the primary immunogenic antigen of the influenza virus, and the D1 H1-17/H3-14 antibody which has been shown to confer potent protection to lethal infection in mice despite lacking neutralization activity. Comparison of sequence coverages upon UV photoactivation of HA and of the HA·antibody complex indicates the elimination of some sequence ions that originate from backbone cleavages exclusively along the putative epitope regions of HA in the presence of the antibody. Mapping the number of sequence ions covering the HA antigen versus the HA·antibody complex highlights regions with suppressed backbone cleavage and allows elucidation of unknown epitopes. Moreover, examining the observed fragment ion types generated by UVPD demonstrates a loss in diversity exclusively along the antigenic determinants upon MS/MS of the antibody–antigen complex. UVPD-MS shows promise as a method to rapidly map epitope regions along antibody–antigen complexes as novel antibodies are discovered or developed.