Abstract
Mass spectrometry is frequently used to determine protein complex topology. By combining in-solution and gas-phase dissociation measurements, information can be indirectly inferred about the original composition of the protein complex. Although the mechanisms behind gas-phase complex dissociation are becoming more established, protein complex dissociation is not always predictable. Here, we looked into the effect of the protein subunits pI on complex dissociation. We chose two structurally similar, hexameric protein complexes that consist of a ring of alternating alpha and beta subunits. For one complex, allophycocyanin, the alpha and beta subunits are structurally similar, almost identical in mass, but have distinct pIs. In contrast, the other complex, phycoerythrin, is structural similar to allophycocyanin, yet the subunits have identical pIs. As predicted based on the structural arrangement, dissociation of phycoerythrin resulted in the observation of both the alpha and beta monomeric subunits in the mass spectrometer. However, for allophycocyanin, the results differed dramatically, with only the alpha monomeric subunit being detected upon gas-phase dissociation. Together, the results highlighted the importance of considering the isoelectric points of individual subunits within a protein complex when using tandem mass spectrometry data to elucidate protein complex topology.
Highlights
T housands of proteins assemble into functional protein complexes to carry out their biological role
Allophycocyanin and phycoerythrin (5–12 μM) were buffer exchanged into 100 mM ammonium acetate solution by use of a 10,000 MWCO centrifugal filter (Amicon)
Upon in-solution dissociation of allophycocyanin by decreasing the pH to 2.5, the beta subunit of allophycocyanin was clearly visible and dominated the mass spectrum (Figure 3c) proving that it is present in solution and in the intact complex
Summary
T housands of proteins assemble into functional protein complexes to carry out their biological role. Deciphering how all these subunits interact within these complexes is a major aim for structural biologists. Tools are continuously being developed to understand the subunit topology within protein complexes and dynamics that are displayed within these macromolecular assemblies. Mass spectrometry is being increasingly used in this area. Using native mass spectrometry techniques, protein complexes can be preserved into the gas phase for structural analysis [1–5].
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