Top-Down Analysis of Disulfide-Linked Proteins Using Photoinduced Radical Reactions and ET-DDC

International Journal of Mass Spectrometry
Sarju AdhikariScott A McLuckey

Abstract

Top-down characterization of proteins via tandem mass spectrometry (MS/MS) can be challenging due to the presence of multiple disulfide bond linkages; which significantly inhibit the backbone cleavage efficiency for the formation of structurally informative fragment ions. In this study, we present a strategy of pairing a solution-phase photoinitiating system with dipolar direct current induced collisional activation of electron transfer products (ET-DDC) of proteins for a top-down MS/MS approach. The photoinitiating system allows for a rapid scission of all the disulfide linkages in the protein (on the time scale of seconds) with high efficiency (near to complete reduction); while ET-DDC collisional activation improves the fragmentation efficiency for the protein via broadband activation of all the first-generation charge reduced precursor ions (e.g., electron transfer no-dissociation or ETnoD products) from electron transfer reactions over a wide mass-to-charge range. As a result, this approach enabled the generation of extensive sequence informative fragment ion yields for a rapid and enhanced structural characterization of disulfide-linked proteins.

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