4.8 Article

Atomistic Insights into the Formation of Nonspecific Protein Complexes during Electrospray Ionization

Journal

ANALYTICAL CHEMISTRY
Volume 93, Issue 37, Pages 12748-12757

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c02836

Keywords

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Funding

  1. Natural Sciences and Engineering Research Council of Canada [RGPIN-2018-04243]

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Native electrospray ionization (ESI)-mass spectrometry (MS) is commonly used for detecting and characterizing multi-protein complexes, however, nonspecific protein clustering in the ESI plume can distort results. This study combines experiments and molecular dynamics simulations to reveal insights into ESI clustering of proteins, demonstrating the formation of nonspecific protein clusters via solvent evaporation. Through MD simulations, the study confirms the viability of charged residue model (CRM) and ion evaporation model (IEM) pathways for protein clustering.
Native electrospray ionization (ESI)-mass spectrometry (MS) is widely used for the detection and characterization of multi-protein complexes. A well-known problem with this approach is the possible occurrence of nonspecific protein clustering in the ESI plume. This effect can distort the results of binding affinity measurements, and it can even generate gas-phase complexes from proteins that are strictly monomeric in bulk solution. By combining experiments and molecular dynamics (MD) simulations, the current work for the first time provides detailed insights into the ESI clustering of proteins. Using ubiquitin as a model system, we demonstrate how the entrapment of more than one protein molecule in an ESI droplet can generate nonspecific clusters (e.g., dimers or trimers) via solvent evaporation to dryness. These events are in line with earlier proposals, according to which protein clustering is associated with the charged residue model (CRM). MD simulations on cytochrome c (which carries a large intrinsic positive charge) confirmed the viability of this CRM avenue. In addition, the cytochrome c data uncovered an alternative mechanism where protein-protein contacts were formed early within ESI droplets, followed by cluster ejection from the droplet surface. This second pathway is consistent with the ion evaporation model (IEM). The observation of these IEM events for large protein clusters is unexpected because the IEM has been thought to be associated primarily with low-molecular-weight analytes. In all cases, our MD simulations produced protein clusters that were stabilized by intermolecular salt bridges. The MD-generated charge states agreed with experiments. Overall, this work reveals that ESI-induced protein clustering does not follow a tightly orchestrated pathway but can proceed along different avenues.

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