Controlling release, unfolding and dissociation of membrane protein complexes in the gas phase through collisional cooling

Abstract

Mass spectrometry of intact membrane protein complexes requires removal of the detergent micelle by collisional activation. We demonstrate that the necessary energy can be obtained by adjusting the degree of collisional cooling in the ion source. This enables us to extend the energy regime for dissociation of membrane protein complexes.Detergents and lipids protect membrane protein complexes during the transition from solution to the gas phase. [1][2][3][4] To enable the study of intact membrane proteins by mass spectrometry, this protective layer has to be removed through collisional activation. This is usually achieved by raising the collision voltage in the ion trap or the collision cell of the instrument (Fig. 1A). However, most detergents require significant amounts of energy to dissociate, 5 and the release of membrane proteins from lipid environments has to date only been achieved using customized high-energy mass spectrometers. 6 Only limited energy can be applied once micelle removal is achieved, which often precludes collision-induced dissociation studies of membrane proteins that can provide valuable information about complex stoichiometry and stability. 7 Ions can acquire energies in the electronvolt range during the passage from atmospheric to low pressure in the source of the mass spectrometer. 8 Maintaining a high pressure at the first pumping stage of the instrument (i.e. the source or "backing" pressure) between the sample cone and extractor cone increases the amount of low-energy collisions and reduces ion velocity (see Supplementary methods, ESI †). As a result, the ions avoid collisional activation and their trajectories are focused to facilitate efficient transmission. [9][10][11][12][13] This process, termed "collisional cooling", is widely used for native MS of protein complexes. This raises the question as to whether reduced collisional cooling could be used to facilitate more efficient detergent removal. We find that the high-energy collisions at low source pressure provide a similar level of activation as achieved in the collision cell. By controlling Carol.Robinson@chem.ox.ac.uk. † Electronic supplementary information (ESI) available: Supplementary methods and Fig. S1. See

Native MS Analysis of Bacteriorhodopsin and an Empty Nanodisc by Orthogonal Acceleration Time-of-Flight, Orbitrap and Ion Cyclotron Resonance

“…It should also be noted that the recorded source pressures on the Orbitrap and FT-ICR (both approximately 2 mbar) instruments are lower than that of the Q-ToF (6 mbar). Landreh 30 recently demonstrated the effects of source backing pressure (Q-ToF only) on membrane protein liberation from a detergent micelle. Lippens et al 31 have also demonstrated this temperature effect for nucleic acids.…”

Native mass spectrometry—A valuable tool in structural biology

“…To overcome this problem, membrane proteins or protein complexes are released from the detergent micelle by CID in the gas phase of the mass spectrometer 89,90 (Figure 4C). A similar effect is observed by reducing collisional cooling in the ion source 91 . However, the required energy to release the intact membrane proteins from the detergent micelle is often too high causing unfolding of the proteins or dissociation of protein subunits from the intact assemblies.…”

“…A similar effect is observed by reducing collisional cooling in the ion source. 91 However, the required energy to release the intact membrane proteins from the detergent micelle is often too high causing unfolding of the proteins or dissociation of protein subunits from the intact assemblies. Screening a variety of MS-compatible detergents followed by fine-tuning of instrument parameters is therefore recommended to determine ideal conditions for native MS experiments of membrane proteins.…”

Electrospray ionization of native membrane proteins proceeds via a charge equilibration step

“…For the mass measurement of these proteins, the micelle is removed through collisional activation inside the mass spectrometer leaving behind the intact protein complex. 17,18 This process implies that the natively membraneembedded regions of the membrane protein's surface area (SA) are covered by detergent molecules during ionization. Ergo, the charge that membrane proteins could potentially acquire should depend on, and be best predicted by, the size of the full complex including the additional size and mass of the detergent micelle.…”