Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry

Abstract

Tandem mass spectrometry of denatured, multiply charged high mass protein precursor ions yield extremely dense spectra with hundreds of broad and overlapping product ion isotopic distributions of differing charge states that yield an elevated baseline of unresolved “noise” centered about the precursor ion. Development of mass analyzers and signal processing methods to increase mass resolving power and manipulation of precursor and product ion charge through solution additives or ion–ion reactions have been thoroughly explored as solutions to spectral congestion. Here, we demonstrate the utility of electron capture dissociation (ECD) coupled with high-resolution cyclic ion mobility spectrometry (cIMS) to greatly increase top-down protein characterization capabilities. Congestion of protein ECD spectra was reduced using cIMS of the ECD product ions and “mobility fractions”, that is, extracted mass spectra for segments of the 2D mobiligram (m/z versus drift time). For small proteins, such as ubiquitin (8.6 kDa), where mass resolving power was not the limiting factor for characterization, pre-IMS ECD and mobility fractions did not significantly increase protein sequence coverage, but an increase in the number of identified product ions was observed. However, a dramatic increase in performance, measured by protein sequence coverage, was observed for larger and more highly charged species, such as the +35 charge state of carbonic anhydrase (29 kDa). Pre-IMS ECD combined with mobility fractions yielded a 135% increase in the number of annotated isotope clusters and a 75% increase in unique product ions compared to processing without using the IMS dimension. These results yielded 89% sequence coverage for carbonic anhydrase.

Ion Mobility Mass Spectrometry (IM-MS) for Structural Biology: Insights Gained by Measuring Mass, Charge, and Collision Cross Section

“…The limitation of these methods are that ETD/ECD is most effective for precursor cation of high charge states, which limits its deployment to native protein complexes that tend to be produced with relatively low charge state distributions. Until recently ECD was only possible on FT-ICR instruments, which also limited its uptake, but now an efficient ECD cell can be retrofitted to both QToF and orbitraps. − Much of the usage of these electron mediated methods has been for peptidecentric studies to provide complementary fragment information to that achieved by CID, however, in native mass spectrometry approaches, they have had more selective application for highly detailed structural investigations for both proteins ,,,− and other biopolymers. − As with UVPD, ECD and ETD provide an opportunity to examine which parts of a given protein sequence are released from the main fold following activation or natively within dynamic complexes, such studies are often well supported with IM-MS measurements, and here we highlight some pertinent examples.…”

Ion Mobility Mass Spectrometry for the Separation and Characterization of Small Molecules

“…The Vion series moved the ion mobility tube and its frontally connected collision cell to the front of the quadrupole. For compound structure interpretation, in addition to the collision-induced dissociation (CID), TWIMS can also combine surface-induced dissociation (SID) or electron capture dissociation, which is essential for revealing ligand-bound and unbound structural information. , Compared to DTIMS, the resolution of TWIMS is not significantly improved (less than 100), which is more suitable for the separation of macromolecules. However, cyclic TWIMS can selectively release ions and further separate the remaining ions, achieving a resolution of several hundred and significantly improving the separation and characterization of more small molecules …”

De novo glycan sequencing by electronic excitation dissociation MS2-guided MS3 analysis on an Omnitrap-Orbitrap hybrid instrument

“…Recently, electromagnetostatic cells that may be installed in non-ICR instruments have become commercially available, allowing protein sequencing by electron-based dissociation on Orbitrap, time-of-ight (TOF), and ion trap mass spectrometers. [42][43][44][45][46][47][48] However, effective ExD characterization of glycans on these instruments has yet to be fully realized, in part because ion-electron interaction occurs during analyte transfer through the ExD cell without trapping, and this limits the ExD efficiency, particularly for higher-energy ExD processes. Improved electron activated dissociation (EAD) efficiency may be achieved by trapping the precursor ions for more extensive interactions with electrons.…”