Molecular Simulation-Based Structural Prediction of Protein Complexes in Mass Spectrometry: The Human Insulin Dimer

Protein electrospray ionization (ESI) mass spectrometry (MS)-based techniques are widely used to provide insight into structural proteomics under the assumption that non-covalent protein complexes being transferred into the gas phase preserve basically the same intermolecular interactions as in solution. Here we investigate the applicability of this assumption by extending our previous structural prediction protocol for single proteins in ESI-MS to protein complexes. We apply our protocol to the human insulin dimer (hIns2) as a test case. Our calculations reproduce the main charge and the collision cross section (CCS) measured in ESI-MS experiments. Molecular dynamics simulations for 0.075 ms show that the complex maximizes intermolecular non-bonded interactions relative to the structure in water, without affecting the cross section. The overall gas-phase structure of hIns2 does exhibit differences with the one in aqueous solution, not inferable from a comparison with calculated CCS. Hence, care should be exerted when interpreting ESI-MS proteomics data based solely on NMR and/or X-ray structural information.Author Summary: Electrospray ionization (ESI) mass spectrometry (MS) plays a pivotal role in proteomics and structural biology. The applications of ESI-MS to protein complexes make use of the assumption that the vaporization of protein complexes into the gas phase (as occurs during ESI-MS) preserves the structural determinants of the complexes that are observed in water. We used computational methods to investigate this key issue by studying the gaseous structure of a pharmacologically relevant protein complex. The complex in the gas phase differs in a subtle yet significant way from the solution structure. This finding is likely of general relevance for protein-protein complexes. Hence, our work implies that the assumption used in proteomic studies, i.e. that in the gas phase non-covalent complexes generally preserve the representative structural determinants observed in the aqueous phase, needs to be reconsidered. Therefore we suggest that the analysis of complexes should be performed on an individual base rather than by generalized principles.