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

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.

Figure 1.

(a) Schematic representation of the Synapt G2-Si instrument used in this study. The inset shows a photo of the EMS cell mounted between the IM and transfer cell. (b) Schematic of the EMS cell, highlighting the path of the protein ions (red arrows) and confined electrons (in blue).

Figure 2.

(a) Charge state series for the native ADH tetramer after online SEC. Dashed lines indicate the calculated maxima (i.e., most abundant isotope peaks) for charge states from 23+ to 28+. (b) Zoom on the 26+ charge state, showing the extreme (for native MS) resolving power and mass accuracy achieved with this method. “Δ” denotes the difference between the observed (blue) and calculated (red) maximum. Note that the Zn²⁺ binding adds 61.91 Da per metal cation (495.32 Da for all eight or approximately 0.34% of the mass of the complex), corresponding to 2.38 m/z at this charge state (19.05 m/z in total), and is thus easily observable with this method. No smoothing of the spectrum was performed.

Figure 3.

(a) Top-down ECD spectrum of 6+ ubiquitin using the electromagnetostatic cell on the Synapt G2-Si, resulting in the cleavage of 62 out of 75 inter-residue bonds (83% cleavage coverage). (b) ECD of 4+ mellitin, resulting in the cleavage of 24 out of 25 bonds (96%). Cleavage diagrams are displayed below the spectra. Note the 30- and 40-fold magnification used; while fragments are very low in intensity compared to the (charge-reduced) precursor, signal-to-noise for these ions is excellent. Charge-reduced species in both panels are annotated as n-radicals for simplicity, although they are actually a complex mixture of ions produced by nondissociative electron capture with and without subsequent loss of H^•, as explored in-depth in previous work.^,

Figure 4.

(a) Native top-down ECD spectrum of 26+ ADH tetramer using the electromagnetostatic cell on the Synapt G2-Si, resulting in the cleavage of 40 out of 346 inter-residue bonds (11.6% cleavage coverage). (b) Native top-down ETD of the same precursor on the same instrument, resulting in the cleavage of 34 bonds (9.8%). Cleavage diagrams are displayed below the spectra (only the first 80 N-terminal residues are shown for clarity). Results from both ETD and ECD are highlighted on the ADH crystal structure in (c) with sequence regions probed by both methods shown in blue and regions probed only with ECD, in red.

Figure 5.

Native top-down IM-ECD spectra of human hemoglobin applying a pre-IM activation voltage at the sampling cone of (a) 25 V and (b) 100 V. Cleavage diagrams are displayed below the spectra with only the first 60 N-terminal residues shown for clarity. Cleavage coverage was 25.0% (α-subunit, 25 V cone), 14.5% (β, 25 V), 29.3% (α, 100 V), and 14.5% (β, 100 V). In (c), results are shown on the hemoglobin tetramer crystal structure (left-hand side) with sequence regions probed with low activation energy in blue and additional regions probed using high energy in red. On the right-hand side of (c), arrival time distributions for the tetramer (mobility measurement immediately prior to ECD) are shown with a low (blue) and high (red) cone voltage.