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

Abstract

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.

Lighting Up at High Potential: Effects of Voltage and Emitter Size in Nanoelectrospray Ionization

“…Results from some molecular dynamics simulations indicate that peptides and proteins can be desorbed from charged droplets either as an extended chain or as intact, folded complexes. − Our results indicate that under conditions where ion desorption from the liquid–air interface occurs for Cs + , Rb + , K + , Na + , Li + , and Cu + , desorption of peptides and proteins does not occur consistent with a large negative solvation energy for these ions. Future work with emitters with even smaller tip diameters may shed additional light into ion formation mechanisms in electrospray ionization.…”

Release of nanodiscs from charged nano-droplets in the electrospray ionization revealed by molecular dynamics simulations

“…However, on one hand, the release of nanodisc from the nano-droplet obtained from the MD simulation at high temperature is consistent with that at room temperature (300 K). On the other hand, in reported studies, MD simulations at high temperature produced the same m/z ratios as the mass spectrometry experiments 21 , 28 , 29 , and the force field for the liquid phase is suitable for modeling gaseous ions 56 . Therefore, the limitations of the methods used in this work may affect the accuracy of the predicted m/z ratio, but likely have little influence on the observed ESI process.…”

“…Therefore, theoretical approaches have been used to extract the relation between charge state and the mass/shape of the gaseous ions from the available mass spectrometry results 10 – 13 . Computational methods, especially molecular dynamics (MD) simulations 14 , 15 , have also been performed to investigate the charge-induced conformational transitions of proteins 16 – 18 and polymers 19 in vacuum, the relation of charge states versus nano-droplet morphologies 20 , and release of analyte ions, such as small ions 21 – 23 , peptides 24 , polymers 25 – 27 , global proteins 28 , and protein complexes 29 , from charged nano-droplets 30 . Here, we explored the spatio-temporal evolution of charged nano-droplets with empty (without protein) lipid nanodiscs at atomistic resolution using MD simulations.…”

Release of lipid nanodiscs from charged nano-droplets in the electrospray ionization process revealed by microsecond atomistic molecular dynamics simulations

“…Therefore, theoretical approaches have been used to extract the relation between charge state and the mass/shape of the gaseous ions from the available mass spectrometry results [9][10][11][12] . Computational methods, especially molecular dynamics (MD) simulations [13][14] , have also been performed to investigate the charge-induced conformational transitions of proteins [15][16][17] and polymers 18 in vacuum, the relation of charge states versus nano-droplet morphologies 19 , and release of analyte ions, such as small ions [20][21][22] , peptides 23 , polymers [24][25][26] , global proteins 27 , and protein complexes 28 , from charged nanodroplets 29 . Here, we explored the spatio-temporal evolution of charged nano-droplets with empty (without protein) lipid nanodiscs at atomistic resolution using MD simulations.…”