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.
Keywords: Collision-induced dissociation; Isoelectric point; Native mass spectrometry; Phycobiliproteins; Protein complex dissociation.