Native electrospray ionization and electron-capture dissociation for comparison of protein structure in solution and the gas phase

Abstract

The importance of protein and protein-complex structure motivates improvements in speed and sensitivity of structure determination in the gas phase and comparison with that in solution or solid state. An opportunity for the gas phase measurement is mass spectrometry (MS) combined with native electrospray ionization (ESI), which delivers large proteins and protein complexes in their near-native states to the gas phase. In this communication, we describe the combination of native ESI, electron-capture dissociation (ECD), and top-down MS for exploring the structures of ubiquitin and cytochrome c in the gas phase and their relation to those in the solid-state and solution. We probe structure by comparing the protein's flexible regions, as predicted by the B-factor in X-ray crystallography, with the ECD fragments. The underlying hypothesis is that maintenance of structure gives fragments that can be predicted from B-factors. This strategy may be applicable in general when X-ray structures are available and extendable to the study of intrinsically disordered proteins.

Conformational Space and Stability of ETD Charge Reduction Products of Ubiquitin

“…This is in excellent agreement with results which were reported recently by Vachet and colleagues [37], who performed ETD in a quadrupole ion trap. This phenomenon could perhaps even provide insight into why, using ECD on FTICR instruments (somewhat harsh interface, ion residence time up to seconds), a rearrangement to a compact, salt-bridge stabilized, yet distinctly non-native structure is consistently reported when spraying ubiquitin from either acidified water/methanol solution [38], or native-like aqueous ammonium acetate [39].…”

Determination of thermodynamic and kinetic properties of biomolecules by mass spectrometry

“…The principle is that if proteins assume a new conformation in the GP, surface-exposed regions will change and top-down ECD will show a fragmentation pattern that will be different than that predicted by SP methods. Ubiquitin and cytochrome C was studied by native ESI-ECD, and top-down MS [22]. Solution NMR and X-ray crystallography models of ubiquitin indicate a flexible C-terminus and a compact Nterminus.…”

Extensive Charge Reduction and Dissociation of Intact Protein Complexes Following Electron Transfer on a Quadrupole-Ion Mobility-Time-of-Flight MS

“…In these experiments, charge reduction and the subsequent transmission and detection of the resulting extremely high m/z species typically require instrument modifications [15,18]. Limited charge reduction is also typically observed as a side reaction in electron capture (ECD) and electron transfer dissociation (ETD) experiments, without instrument modification, particularly when working with intact proteins [19][20][21][22][23][24]. In addition, ECD or ETD fragments can remain bound to each other by noncovalent interactions so that they appear as chargereduced precursor ions in the spectra [24][25][26][27].…”