Immunoglobulins represent critical protein constructs in the dual contexts of human disease and biotechnology.  In both areas, these proteins present significant analytical challenges in their rapid characterization.  Specifically, subtle deviations in the structure and/or sequence of the protein domains present can greatly alter the specificity of a given antibody/antigen interaction.  Since intact antibodies are possessed of, large (~150 kDa), multi-domain, disulfide bounded architectures, such structural changes can manifest in many ways and are often difficult to detect.  We constructed protocols for the analysis of intact antibodies based on ion mobility-mass spectrometry (IM-MS).  IM separates intact antibody ions generated by nano-electrospray ionization (nESI) on the millisecond timescale according to their orientationally-averaged size and charge.  The technology works by injecting packets of ions into an ion guide pressurized with inert neutrals, and using a pulsed electric field to drive the ions towards an exit aperture.  Larger, less charged ions will take longer to migrate through the IM separator than smaller ions possessed of larger charge states.   Our protocol for the analysis of intact antibodies involves the collisional heating of trapped antibody ions within the instrument, followed by IM-MS analysis of the activated antibody ions.  After sufficient collisional heating, antibody ions undergo unfolding in the gas-phase.  Our IM-MS experiments record the unfolding pathways observed for these ions in detail, and our protocol endeavours to build fingerprints based on such collision induced unfolding (CIU) data that can be used to differentiate the number and pattern of disulfide bonds within intact antibody structures.  This presentation will contain our latest data validating our approach, and point the future use of IM-MS and CIU in rapid quality control applications for intact antibody preparations.

Collision induced unfolding of intact antibody ions in the gas phase: A new method for rapidly characterizing disulfide bonding patterns

248th National Meeting (2014)

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