Analysis of Oxidatively Damaged Proteins by Mass Spectrometry
Abstract
As humans age, exposure to oxidative stress may induce protein degradation or aggregation; both resulting in loss of protein function. Protein oxidative damage remains a dominant pathology in many common ailments. To combat these pathologies, scientists must understand the nature of oxidative modifications and their effects on protein structure and dynamics. This work employs a range of mass spectrometry (MS) methods to characterize and analyze the effects of oxidative damage on the model protein myoglobin (Mb). Mb was oxidized using tert-butyl hydroperoxide, and the resulting modifications were characterized by top-down and bottom-up MS workflows. Hydrogen/deuterium exchange MS indicated elevated structural dynamics in oxidatively modified regions. Collision-induced activation showed that oxidized Mb loses heme more readily than its unmodified counterpart. Surprisingly, ion mobility experiments uncovered that collision-induced unfolding produces more compact non-native gas phase structures for the oxidized protein. The methods applied provide an analytical foundation for the comprehensive characterization of oxidative damage that will be applicable to many other proteins.