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Anal Chem. 2020 Mar 3;92(5):3674-3681. doi: 10.1021/acs.analchem.9b04763. Epub 2020 Feb 11.

Top-Down Characterization of Denatured Proteins and Native Protein Complexes Using Electron Capture Dissociation Implemented within a Modified Ion Mobility-Mass Spectrometer.

Author information

1
Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow SK9 4AX, United Kingdom.
2
e-MSion Inc., 2121 NE Jack London Drive, Corvallis, Oregon 97330, United States.
3
Linus Pauling Institute and the Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States.
4
School of Engineering, University of Warwick, Coventry CV4 7AL, United Kingdom.
5
Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom.

Abstract

Electron-based fragmentation methods have revolutionized biomolecular mass spectrometry, in particular native and top-down protein analysis. Here, we report the use of a new electromagnetostatic cell to perform electron capture dissociation (ECD) within a quadrupole/ion mobility/time-of-flight mass spectrometer. This cell was installed between the ion mobility and time-of-flight regions of the instrument, and fragmentation was fast enough to be compatible with mobility separation. The instrument was already fitted with electron transfer dissociation (ETD) between the quadrupole and mobility regions prior to modification. We show excellent fragmentation efficiency for denatured peptides and proteins without the need to trap ions in the gas phase. Additionally, we demonstrate native top-down backbone fragmentation of noncovalent protein complexes, leading to comparable sequence coverage to what was achieved using the instrument's existing ETD capabilities. Limited collisional ion activation of the hemoglobin tetramer before ECD was reflected in the observed fragmentation pattern, and complementary ion mobility measurements prior to ECD provided orthogonal evidence of monomer unfolding within this complex. The approach demonstrated here provides a powerful platform for both top-down proteomics and mass spectrometry-based structural biology studies.

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