Hydrogen deuterium exchange mass spectrometry identifies the dominant paratope in CD20 antigen binding to the NCD1.2 monoclonal antibody.

A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.

The use of ion mobility mass spectrometry to assist protein design: a case study on zinc finger fold versus coiled coil interactions.

The dramatic conformational change in zinc fingers on binding metal ions for DNA recognition makes their structure-function behaviour an attractive target to mimic in de novo designed peptides. Mass spectrometry, with its high throughput and low sample consumption provides insight into how primary amino acid sequence can encode stable tertiary fold. We present here the use of ion mobility mass spectrometry (IM-MS) coupled with molecular dynamics (MD) simulations as a rapid analytical platform to inform de novo design efforts for peptide-metal and peptide-peptide interactions. A dual peptide-based synthetic system, ZiCop based on a zinc finger peptide motif, and a coiled coil partner peptide Pp, have been investigated. Titration mass spectrometry determines the relative binding affinities of different divalent metal ions as Zn(2+) > Co(2+) ≫ Ca(2+). With collision induced dissociation (CID), we probe complex stability, and establish that peptide-metal interactions are stronger and more 'specific' than those of peptide-peptide complexes, and the anticipated hetero-dimeric complex is more stable than the two homo-dimers. Collision cross-sections (CCS) measurements by IM-MS reveal increased stability with respect to unfolding of the metal-bound peptide over its apo-form, and further, larger collision cross sections for the hetero-dimeric forms suggest that dimeric species formed in the absence of metal are coiled coil like. MD supports these structural assignments, backed up by data from visible light absorbance measurements.