A Triaxial Probe for On-line Proteolysis Coupled with Hydrogen/Deuterium Exchange-Electrospray Mass Spectrometry

An on-line proteolysis system utilizing a triaxial electrospray probe was developed to aid localization of the hydrogen-bonding interaction sites in hydrogen/deuterium exchange-mass spectrometry (HDX-MS) studies of Aβ (1-40) fibrils. The probe allows delayed introduction of the organic solvent component needed for stable electrospray, thus enhancing hydrolysis performance relative to that of a coaxial probe. Effective on-line digestion was accomplished in ∼12 s. The probe should be of general utility for HDX-MS studies of amyloid fibrils and other protein aggregates.