Radical solutions: Principles and application of electron-based dissociation in mass spectrometry-based analysis of protein structure

Abstract

In recent years, electron capture (ECD) and electron transfer dissociation (ETD) have emerged as two of the most useful methods in mass spectrometry-based protein analysis, evidenced by a considerable and growing body of literature. In large part, the interest in these methods is due to their ability to induce backbone fragmentation with very little disruption of noncovalent interactions which allows inference of information regarding higher order structure from the observed fragmentation behavior. Here, we review the evolution of electron-based dissociation methods, and pay particular attention to their application in "native" mass spectrometry, their mechanism, determinants of fragmentation behavior, and recent developments in available instrumentation. Although we focus on the two most widely used methods-ECD and ETD-we also discuss the use of other ion/electron, ion/ion, and ion/neutral fragmentation methods, useful for interrogation of a range of classes of biomolecules in positive- and negative-ion mode, and speculate about how this exciting field might evolve in the coming years.

Keywords: electron capture dissociation; electron transfer dissociation; noncovalent complex; protein folding; top-down fragmentation.

Figure 1.

Cornell and Utah-Washington mechanism for ECD.

Figure 2.

Timeline of ‘milestones’ in the development of ECD and ETD.

Figure 3.

Selected implementations of ECD and ETD. (A) ECD on FTICR; (B) atmospheric pressure ECD (Waters); (C) electromagnetostatic (EMS) cell; (D) ETD on 3D ion trap (e.g. Bruker AmaZon); (E) ETD on LTQ/Orbitrap; (F) ETD within T-wave device (e.g. Waters Synapt); (G) ETD on QTOF (e.g. Bruker maXis). Abbreviations used: UV – ultraviolet; CI – chemical ionization; LTQ – linear trap quadrupole; GD – glow discharge.

Figure 4.

Periodic product-ion abundance in the ECD spectrum of a 3+ α-helical transmembrane domain of the influenza virus A membrane protein M2. Adapted with permission from (Ben Hamidane, et al., 2009b).

Figure 5.

ECD as a structural probe for different ubiquitin charge states. Fragment intensities per cleavage site for charge states between 6+ and 13+ are shown on the left (black segments – cfragments, open segments – z-fragments, grey fragments – a- and y-fragments), whereas the righthand side shows proposed gas-phase structures with salt bridge patterns that account for regions that display low or no dissociation. Adapted with permission from (Breuker, et al., 2002) (Copyright (2002) American Chemical Society) and (Oh, et al., 2002) (Copyright (2002) National Academy of Sciences).

Figure 6.

Stepwise evolution after ESI of the structure of a globular protein (e.g., cytochrome c, ubiquitin). (A) Native protein covered with a monolayer of H₂O, followed by (B) nanosecond H₂O loss and concomitant cooling. (C) Exterior ionic functionalities lose hydration and rapidly (about 10 picoseconds) collapse. (D) The exterior-collapsed “near-native” protein, subsequently undergoes thermal re-equilibration, via (E) millisecond loss of hydrophobic bonding, and (F) millisecond loss of electrostatic interactions. (G) Formation of new noncovalent bonds occurs in seconds, and ultimately leads to stabilization to conformers that represent gas-phase energy minima in minutes. Reprinted with permission from (Breuker& McLafferty, 2008) (Copyright (2008) National Academy of Sciences).

Figure 7.

Fragmentation patterns observed in ECD and ETD of the native ADH tetramer. (A) FTICRECD with the entire native charge state distribution subjected to ECD; (B) QTOF-ETD of the 26+ tetramer with varying degrees of pre-ETD collisional activation and no supplemental activation (only additional fragments colored for higher cone voltages, i.e., fragments observed with 40 or 80 V preheating are generally also found with 120 V); (C), as in (B), but with minimal pre-ETD voltages and 70 V of supplemental activation. Panel (A) is based on data published in (Zhang, et al., 2011a) (Copyright (2011) American Chemical Society); data to generate panels (B) and (C) is found in (Lermyte& Sobott, 2015). Crystallographic B factor is shown on the vertical axis. Horizontal axis only shows the first 70 (of 347) N-terminal residues for clarity (no fragments from further along the sequence were observed). Panels on the right shown the corresponding ExD spectra (note the 10fold magnification of the region that contains ETD fragments in Panel C), with the result of 120 V of pre-heating shown in Panel B.

Figure 8.

Crystal structure of the ADH tetramer (Protein Data Bank accession code 4W6Z) with ETD cleavage sites observed with minimal pre-ETD voltages and 70 V of supplemental activation (as in Figure 7C) shown in red. Inset shows how cleavage near charge sites (mostly found on the exposed surface) is expected in both the Cornell and Utah-Washington mechanisms for ExD.