Integrated Top-Down and Bottom-Up Mass Spectrometry for Characterization of Diselenide Bridging Patterns of Synthetic Selenoproteins

With the rapid acceleration in the design and development
of new
biotherapeutics, ensuring consistent quality and understanding degradation
pathways remain paramount, requiring an array of analytical methods
including mass spectrometry. The incorporation of non-canonical amino
acids, such as for synthetic selenoproteins, creates additional challenges.
A comprehensive strategy to characterize selenoproteins should serve
dual purposes of providing sequence confirmation and mapping of selenocysteine
bridge locations and the identification of unanticipated side products.
In the present study, a combined approach exploiting the benefits
of both top-down and bottom-up mass spectrometry was developed. Both
electron-transfer/higher-energy collision dissociation and 213 nm
ultraviolet photodissociation were utilized to provide complementary
information, allowing high quality characterization, localization
of diselenide bridges for complex proteins, and the identification
of previously unreported selenoprotein dimers.