The role of conformation on electron capture dissociation of ubiquitin

Abstract

Effects of protein conformation on electron capture dissociation (ECD) were investigated using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and Fourier-transform ion cyclotron resonance mass spectrometry. Under the conditions of these experiments, the electron capture efficiency of ubiquitin 6ϩ formed from three different solution compositions differs significantly, ranging from 51 Ϯ 7% for ions formed from an acidified water/methanol solution to 88 Ϯ 2% for ions formed from a buffered aqueous solution. This result clearly indicates that these protein ions retain a memory of their solution-phase structure and that conformational differences can be probed in an ECD experiment. Multiple conformers for the 7ϩ and 8ϩ charge states of ubiquitin were separated using FAIMS. ECD spectra of conformer selected ions of the same charge states differ both in electron capture efficiency and in the fragment ion intensities. Conformers of a given charge state that have smaller collisional cross sections can have either a larger or smaller electron capture efficiency. A greater electron capture efficiency was observed for ubiquitin 6ϩ that has the same collisional cross section as one ubiquitin 7ϩ conformer, despite the lower charge state. These results indicate that the shape of the molecule can have a greater effect on electron capture efficiency than either collisional cross section or charge state alone. The cleavage locations of different conformers of a given charge state were the same indicating that the presence of different conformers in the gas phase is not due to difference in where charges are located, but rather reflect conformational differences most likely originating from solution. Small neutral losses observed from the singly-and doubly-reduced ubiquitin 6ϩ do not show a temperature dependence to their formation, consistent with these ions being formed by nonergodic processes. [7][8][9] can be obtained. Methods based on this electron capture process appear promising for bottom up and top down approaches in proteomics due to the high information content obtained in the fragmentation spectra [10 -12]. Sequence coverage of over 90% has been reported for small proteins [13]. Several methods for implementing electron capture that use different electron sources have been reported: electron capture dissociation (ECD) with free electrons from a heated filament or cathode [1-3], electron-transfer dissociation utilizing anion reagents [14 -17], electron removal by electron impact followed by capture of a free electron in electronic excitation dissociation [18], and collisionally induced electron-transfer from neutral atoms or molecules [19,20].A consequence of the extensive fragmentation produced by ECD is that the precursor ion population can be fractionated into potentially hundreds or even thousands of fragment ions. Thus, the intensity of any particular fragment ion is typically small compared with the original precursor ion intensity [1]. High electron capture and fragmentation efficiency are cr...

Nonergodicity in electron capture dissociation investigated using hydrated ion nanocalorimetry

“…A key distinction between these experiments is that the dissociation processes for the hydrated divalent metal ions are clearly different from those of peptides and proteins. Loss of water molecules resulting from ECD of proteins has been observed [31], but this is a minor process. Product ions corresponding to the loss of a H atom can be the dominant process observed for ECID of diprotonated dipeptides [32], but loss of a H atom is less significant for larger peptides.…”

Elucidating the site of protein-ATP binding by top-down mass spectrometry

“…It is not clear if a ligand-induced solution-phase conformational change suggested for ATP-binding by AK [27,41,42] is affecting the dissociation behavior of the gas-phase AK-ATP complex, i.e., CAD efficiencies are significantly reduced for the ATP-bound protein, following the conversion from an open to closed conformation with ATP binding [41,42]. Based on previous work by others, including McLafferty [43], O'Connor [44], and Williams [45], it should be possible to probe differences in protein conformation by ECD. However, from our data for AK and the AK-ATP complex, aiECD efficiencies appear to depend more on charge state than conformation; sequence coverage and the number of products increases from 31% and 16% for the 9ϩ apo-AK, respectively, to 79% and 41% for the 10ϩ charge state.…”

Measuring the extent and width of internal energy deposition in ion activation using nanocalorimetry

“…These results provide additional evidence for the accuracy with which condensed phase thermochemical values can be deduced from gaseous nanocalorimetry experiments. (J Am Soc Mass Spectrom 2010, 21, 615-625) © 2010 American Society for Mass Spectrometry C apture of a low-energy electron by a multiply charged peptide or protein can result in extensive and relatively non-selective fragmentation of the amide backbone, from which information about the amino acid sequence [1-3], post-translational modification sites [3][4][5], and higher-order structure [2,6,7] can be obtained. Similar fragmentation can be obtained by transferring an electron to the peptide or protein cation from a molecular anion [8] or from atoms [9,10].…”