Optimizing macromolecular tandem mass spectrometry of large non-covalent complexes using heavy collision gases

We evaluated and optimized conditions for tandem mass spectrometry on macromolecular protein complexes, using a modified Q-ToF instrument varying the gas pressure inside the hexapole collision cell, and studied the influence of using different collision gases, e.g. argon, krypton and xenon. These variations affected ion transmission through the instrument and dissociation efficiency In particular the high mass fragment ions were generated more readily and transmitted more efficiently using the heavier xenon as collision gas. We used the 800 kDa GroEL 14-mer chaperone machinery for these initial studies. Applying the optimized tandem mass spectrometry conditions, i.e. relatively high pressures and xenon as collision gas, we studied in detail the influence the binding of one and two gp5 substrate proteins had on the gas-phase dissociation of the chaperonin complex constructing break-down diagrams. We observed that the binding of substrate polypeptides had a significant effect on the gas-phase stability of the chaperonin complex, with the complex containing one and two substrate molecules being less susceptible towards dissociation than the substrate free GroEL. Interestingly, the GroEL:gp5 and GroEL:(gp5)2 complexes dissociate exclusively via the elimination of GroEL monomeric ions, indicating that both gp5 substrates are likely encapsulated inside the two cavities of, respectively, the cis and trans ring of the GroEL. From all data acquired, we conclude that the heavier xenon is the preferred collision gas for tandem mass spectrometry on very large macromolecular complexes.