Protein complexes perform a wide variety of functions within biological systems. Determining the structure of a protein complex can lead to a greater understanding of its function. In their recent publication, Belov et al. suggest that native mass spectrometry (MS) holds great promise, particularly with respect to increased understanding of quaternary and secondary protein structure.1
Although many advances in MS have occurred, such as the development of low-field ion mobility spectrometry alongside MS, the goal now is to extend this technology to characterize composition and stoichiometry of native protein complexes. Using a non-covalent tetramer complex of pyruvate kinase (232 kDa), as well as phosphorylase B (194 kDa) and GroEL (801 kDa) as the models for this investigation, Belov et al. developed a two-step fragmentation approach for large protein complexes. Their successful pseudo-MS3 strategy was made possible by modifications to a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific). The researchers added a dual-funnel interface with orthogonal ion injection. This made it possible to adjust the ion resistance time, which led to an enhanced droplet desolvation. In addition, the researchers also added a bent flatpole with an axial electric field to ensure the ion transmission occurred at a low incoming ion flux. Other modifications included reduced frequencies on the RF multipoles, an increased gas intake and the addition of an image current preamplifier with improved linearity.
The researchers subjected the protein complexes to different degrees of collisional activation. They were able to sequentially dissociate the complexes into highly charged monomers, which were then dissociated to a set of multiply charged fragmentation products. Ultimately, they were able to determine that an increase in the collision energy resulted in dissociation of the protein complex and a release of highly charged monomer species.
The researchers were able to match the signals against the known intact monomers. They identified two subunit backbone fragments from pyruvate kinase, identified at mass measurement accuracies of 11.6 ppm (y45, 4898.72 Da) and 3.3 ppm (b100, 10927.30 Da). From this, a total of 63 unique subunit backbone fragments were identified at a mass resolution of 70,000, corresponding to 40 y- and 23 b-multiply charged fragment ions (PDE score, 11.5; expectation value, 3 × 10–13).
Similarly, the researchers also identified monomers of GroEL. The GroEL protein complex yielded 6 y and 49 b fragment ions with a mass measurement accuracy better than 15 ppm (PDE score, 10.9; expectation value, 4 × 10−23).
Although this two-step fragmentation approach was successful at improving the desolvation, Belov and colleagues suggest that additional experiments and enhancements are necessary. They suggest further that refining the interface to improve spectral assignment at low collision energies, deepening the fragmentation, and improving the desolvation efficiency are all areas worthy of future investigation.
1. Belov, M., et al. (2013) “From Protein Complexes to Subunit Backbone Fragments: A Multi-stage Approach to Native Mass Spectrometry,” Analytical Chemistry, 85(23) (pp. 11163–73), doi: 10.1021/ac4029328.
Post Author: Emily Humphreys. As a biology undergraduate at the University of Utah, Emily balanced a heavy class schedule while working long hours in a lab studying eye development. Following graduation, she became involved in infectious disease and aging research involving SNPS.
While she enjoyed the thrill of research, Emily has since traded bench work for science journalism.
And has been a regular contributor to Accelerating Science since 2012.