Conformational Distribution of Bradykinin [bk + 2 H]²⁺ Revealed by Cold Ion Spectroscopy Coupled with FAIMS

Abstract

We employ cold ion spectroscopy (CIS) in conjunction with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to study the peptide bradykinin in its doubly protonated charge state ([bk+2 H] 2+ ). Using FAIMS, we partially separate the electrosprayed [bk+2 H] 2+ ions into two conformational families and selectively introduce one of them at a time into a cold ion trap mass spectrometer, where we probe them by UV photofragment spectroscopy. Although the two conformational families have distinct electronic spectra, some cross-conformer contamination can be observed under certain conditions. We demonstrate that this contamination comes from isomerization of ions energized during and/or after their separation and not from incomplete separation of the initially electrosprayed conformations in the FAIMS stage. By varying the injection voltage of the ions into our mass spectrometer, we can intentionally induce isomerization to produce what seems to be a gas phase equilibrium distribution of conformers. This distribution is different from the one produced initially by electrospray, indicating that some of the conformers are kinetically trapped and may be related to conformers that are more favored in solution.

Infrared Spectroscopy of Mobility-Selected H+-Gly-Pro-Gly-Gly (GPGG)

“…The trans-Pro species readily isomerizes to cis-Pro when it is energized by collisions; however, under the room temperature conditions of our drift tube, it is still energetically accessible and remains a small portion of the annealed distribution. A similar observation has been made for a variety of other peptidic systems [18,31] and suggests that the relative conformer populations we measure after annealing reflect the Boltzmann weights of these states at a given temperature. The exact temperature to which the relative populations correspond is not obvious; however, it may reflect that of the drift tube (300 K), but it may also reflect a higher temperature, depending on the rate of cooling in the drift tube and the height of the barriers separating the conformers.…”

“…Mass spectrometry and ion mobility would be supplemented by additional structural information, while spectroscopy would be simplified by the presence of mass and conformational filters. Analogous experiments have been developed using UV photofragmentation [30,31], but infrared spectroscopy has the advantage of providing vibrational information that can be compared directly with theoretical calculations.…”

Cryogenic Methods for the Spectroscopy of Large, Biomolecular Ions

“…Although these bands originate from higher energy states of the molecule, they are different from what we normally consider hot bands because the molecule has a different equilibrium geometry and there is a barrier on the potential energy surface separating the conformers. In principle, one could attempt to anneal the molecule by heating and re-cooling to transfer the trapped population into the lowest energy conformation -such annealing has been done both in ion mobility experiments [9,23] and in spectroscopic experiments [24]. However, it may be that these trapped conformations are the most interesting to interrogate.…”

“…One possibility to help deal with this problem is to separate different conformations of the ions physically before injecting them into an ion trap, and one approach to doing this is to combine ion mobility with cold ion spectroscopy. In our laboratory we have recently combined Field Asymmetric Ion Mobility Spectrometry (FAIMS) with a cold, 22-pole ion trap to perform conformer pre-selection prior to ion trapping and UV photofragment spectroscopy [24,142]. Since conformer overlap is a major Returning to where much of the impetus for buffer-gas cooling of ions began, Dieter Gerlich, in collaboration with the Roithova group in Prague, has recently developed a new wire-based quadrupole ion trap, which they inserted into a commercial tandem mass spectrometer (Fig.…”

“…FAIMS uses the difference in ion mobility in a gas at atmospheric pressure at high and low electric fields to deflect different conformational states of a molecule perpendicular to their drift direction. Figure 24 shows a schematic of a commercial FAIMS source (Thermo Fisher) coupled to the inlet of our cold-ion spectrometer [24]. The electrosprayed ions are entrained in nitrogen and drawn into the gap between an inner cylindrical electrode and an outer electrode.…”