Background:
Biotherapeutics, particularly monoclonal antibodies (mAbs), are a maturing
class of drugs capable of treating a wide range of diseases. Therapeutic function and solutionstability
are linked to the proper three-dimensional organization of the primary sequence into
Higher Order Structure (HOS) as well as the timescales of protein motions (dynamics). Methods
that directly monitor protein HOS and dynamics are important for mapping therapeutically relevant
protein-protein interactions and assessing properly folded structures. Irreversible covalent protein
footprinting Mass Spectrometry (MS) tools, such as site-specific amino acid labeling and hydroxyl
radical footprinting are analytical techniques capable of monitoring the side chain solvent accessibility
influenced by tertiary and quaternary structure. Here we discuss the methodology, examples
of biotherapeutic applications, and the future directions of irreversible covalent protein footprinting
MS in biotherapeutic research and development.
Conclusion:
Bottom-up mass spectrometry using irreversible labeling techniques provide valuable
information for characterizing solution-phase protein structure. Examples range from epitope mapping
and protein-ligand interactions, to probing challenging structures of membrane proteins. By
paring these techniques with hydrogen-deuterium exchange, spectroscopic analysis, or static-phase
structural data such as crystallography or electron microscopy, a comprehensive understanding of
protein structure can be obtained.
