Conformational Space and Stability of ETD Charge Reduction Products of Ubiquitin

Abstract

Due to its versatility, electron transfer dissociation (ETD) has become one of the most commonly utilized fragmentation techniques in both native and non-native top-down mass spectrometry. However, several competing reactions primarily different forms of charge reduction occur under ETD conditions, as evidenced by the distorted isotope patterns usually observed. In this work, we analyze these isotope patterns to compare the stability of ETnoD products, specifically noncovalent c/z fragment complexes, across a range of ubiquitin conformational states. Using ion mobility, we find that more extended states are more prone to fragment release. We obtain evidence that for a given charge state, populations of ubiquitin ions formed either directly by electrospray ionization or through collapse of more extended states upon charge reduction, span a similar range of collision cross-sections. Products of gas-phase collapse are however less stabilized towards unfolding than the native conformation, indicating that the ions retain a memory of previous conformational states. Furthermore, this collapse of charge--collisional activation, with possible implications for the kinetics of gas-phase compaction.

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

“…The reaction is implemented within a travelling‐wave ion guide (Figure F) located between both mass analyzers in a hybrid quadrupole/time‐of‐flight instrument (Synapt G2, later also adopted in the G2‐S and G2‐Si models). The ion‐mobility capabilities of this instrument (IM cell located immediately downstream of the ETD cell) allow global structural characterization with an orthogonal method, as well as conformation‐ or charge state‐selective fragmentation, again through dissociation of noncovalent fragment complexes, and interrogation of the structure of charge‐reduced species . Bruker (Bremen, Germany) has also implemented ETD on a quadrupole/time‐of‐flight instrument (maXis HD; Figure G), also by placing the reaction cell between both mass analyzers .…”

“…The ion-mobility capabilities of this instrument (IM cell located immediately downstream of the ETD cell) allow global structural characterization with an orthogonal method, as well as conformation-or charge state-selective fragmentation, again through dissociation of noncovalent fragment complexes, 103 and interrogation of the structure of charge-reduced species. [104][105][106] Bruker (Bremen, Germany) has also implemented ETD on a quadrupole/time-of-flight instrument (maXis HD; Figure 3G), also by placing the reaction cell between both mass analyzers. 107 This implementation routinely achieves a mass resolution of 40 000 or more, with mass accuracy in the low-ppm range, 108 which makes this instrument particularly attractive in a top-down proteomics context.…”

Opposite Structural Effects of Epigallocatechin-3-gallate and Dopamine Binding to α-Synuclein

“…However, such a mechanism would be expected to produce the more extended conformer also in the "empty" protein, by dissociation of the ligand from the 1:1 complex, which is not the case here. Furthermore, structures would be expected to rearrange upon charge reduction 49 . Recently, conformational studies on IDPs by ESI-MS and ESI-IM-MS have been questioned 50 .…”

masstodon: A Tool for Assigning Peaks and Modeling Electron Transfer Reactions in Top-Down Mass Spectrometry

“…These fragmentation methods allow the cleavage of the backbone of a protein or peptide without significantly disrupting other bonds (even preserving noncovalent interactions) and as such, much effort has gone into the use of ExD methods for top-down sequencing, as well as the study of labile posttranslational modifications and even binding sites of non-covalent ligands. [3][4][5][6][7][8][9][10][11][12][13][14] Additionally, considerable efforts have been made to determine preferential reaction pathways and cleavage sites in ExD of known precursors, to obtain insight into gas-phase protein/peptide conformation [15][16][17][18][19][20][21][22][23][24][25] as well as to investigate the reaction mechanism. [26][27][28] Ideally, reaction products are not only identified, but also quantified in these efforts.…”

“…These shifts (by an integer number of hydrogen masses) are commonly observed in ExD spectra and provide information on reaction pathways. 25,35,36 As such, it is desirable to preserve the information contained in observed isotope distributions during and after the deconvolution procedure.…”