Analysis of Chaperone Complexes by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Investigation of methodologies for analyses of noncovalently bound protein assemblies using Fourier transformation ion cyclotron resonance mass spectrometry (FT-ICR-MS) and quadrupole Time-of-Flight (qToF) mass spectrometry. Specifically, the co-chaperonins GroEL and gp31 are used to perform activation measurements on in the gas-phase and in the solution-phase. Both protein complexes are noncovalently bound homoheptamers of 72kDa and 84kDa respectively. They have a slight functional difference which is reflected in a slight structural difference. Using the FT-ICR-MS techniques collisional activated dissociation (CAD), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) the fragmentation pathways of both complexes are investigated. Unexpectedly, ECD leads to fission of noncovalent bonds between the subunits within the complex, resulting in ejection of a monomer from the complex. The ejected monomer remains relatively folded and does not take up many charges form the rest of the complex, indicating that the dissociation process is fast. Different gas-phase conformations lead to different breakdown pathways when using ECD. Collisional activation of the complexes leads to ejection of a highly charged, i.e. unfolded, monomer from the assembly. Various collisional activation techniques with differing timeframes of activation have been used (from fast to relatively slow activation): nozzle-skimmer (NS)-CAD in the source of the mass spectrometer, on resonance CAD in the ICR-cell, q-ToF CAD in the collision cell of the qToF and sustained off-resonance (SORI)-CAD in the ICR cell. The extend of unfolding is dependent on the timeframe of collisional activation. Faster activation leads to an uptake of fewer charges by the monomer compared to slower activation. This indicates that the dissociation process is governed by both the charge redistribution over the complex and the actual collisions transforming the kinetic energy into internal energy. The gas-phase stabilities of GroES and gp31 are comparable when using collisional activation. Solution-phase thermal activation of the complexes is monitored by MS to investigate the fragmentation pathways in solution and compare that to the fragmentation pathway in the gas-phase. Solution-phase thermal manipulation is performed using a custom designed temperature controlled electrospray setup. Using this setup the sample temperature can be accurately controlled up to the moment of electrospray, effectively probing the prevailing chemical/thermal equilibrium. All lower oligomeric stoichiometries of the complexes are observed at certain temperatures when increasing the sample temperature, indicating that the dissociation process from heptamer to monomer cannot be seen as a single-step process. Fluorescence measurements of the thermal activation result in dissociation temperatures that are comparable to the results of the MS measurements. Each subsequent lower oligomer is relatively more unfolded as can be deduced from the higher number of charges per subunit on the lower oligomers. Thermal activation in the gas-phase does not result in appearance of the intermediate oligomeric forms of the protein complexes, indicating that the solvent has a large influence on stabilizing the formation of the intermediate oligomers in solution.