Because integrated commercial packages for data acquisition and analysis are available, HDX-MS has been widely used in biopharmaceutical research.
In HDX studies, electrospray ionization MS detects mass increases from exchanged hydrogen with deuterium. The back-exchange of deuterium to hydrogen occurs at substantial rates, requiring the reaction to be quenched rapidly by cooling the sample to 1°C while suspended in acidic buffer (pH 2.7). The quenching must be promptly followed by peptic digestion and then LC-MS analysis.
While HDX can provide insight into solvent addressable backbone amide hydrogens, its time-course incubation often results in generating products from both rapid and slow exchange processes. Rapid exchange is associated with solvent addressable residues with unbound amide hydrogens, while slower reactions occur with internal, titratable, backbone amide protons involved in hydrogen bonds that make up secondary structural elements, whose exchange rates are driven by conformational change. The net result is an ambiguous understanding of HOS and secondary structure stability.
Performing HDX HOS analysis is a complex and burdensome process. Perhaps this point is best exemplified by Professor Mark Chance of Case Western Reserve University when he remarked, “I invented HRPF because HDX is just too hard to do.”