Hybridizing Ultraviolet Photodissociation with Electron Transfer Dissociation for Intact Protein Characterization

• Published in: Analytical chemistry (Washington) Vol. 86; no. 21; pp. 10970 - 10977
• Main Authors: Cannon, Joe R, Holden, Dustin D, Brodbelt, Jennifer S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 04.11.2014
• Subjects: Chromatography; Dissociation energy; Electron transfer; Electron Transport; Fragmentation; Hybridization; Ions; Liquid chromatography; Mass spectrometers; Mass spectrometry; Photodissociation; Proteins; Proteins - chemistry; Ultraviolet; Ultraviolet Rays
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac5036082

We report a hybrid fragmentation method involving electron transfer dissociation (ETD) combined with ultraviolet photodissociation (UVPD) at 193 nm for analysis of intact proteins in an Orbitrap mass spectrometer. Integrating the two fragmentation methods resulted in an increase in the number of identified c- and z-type ions observed when compared to UVPD or ETD alone, as well as generating a more balanced distribution of a/x, b/y, and c/z ion types. Additionally, the method was shown to decrease spectral congestion via fragmentation of multiple (charge-reduced) precursors. This hybrid activation method was facilitated by performing both ETD and UVPD within the higher energy collisional dissociation (HCD) cell of the Orbitrap mass spectrometer, which afforded an increase in the total number of fragment ions in comparison to the analogous MS3 format in which ETD and UVPD were undertaken in separate segments of the mass spectrometer. The feasibility of the hybrid method for characterization of proteins on a liquid chromatography timescale characterization was demonstrated for intact ribosomal proteins.