Release of Native-like Gaseous Proteins from Electrospray Droplets via the Charged Residue Mechanism: Insights from Molecular Dynamics Simulations

Abstract

The mechanism whereby gaseous protein ions are released from charged solvent droplets during electrospray ionization (ESI) remains a matter of debate. Also, it is unclear to what extent electrosprayed proteins retain their solution structure. Molecular dynamics (MD) simulations offer insights into the temporal evolution of protein systems. Surprisingly, there have been no all-atom simulations of the protein ESI process to date. The current work closes this gap by investigating the behavior of protein-containing aqueous nanodroplets that carry excess positive charge. We focus on "native ESI", where proteins initially adopt their biologically active solution structures. ESI proceeds while the protein remains entrapped within the droplet. Protein release into the gas phase occurs upon solvent evaporation to dryness. Droplet shrinkage is accompanied by ejection of charge carriers (Na(+) for the conditions chosen here), keeping the droplet at ∼85% of the Rayleigh limit throughout its life cycle. Any remaining charge carriers bind to the protein as the final solvent molecules evaporate. The outcome of these events is largely independent of the initial protein charge and the mode of charge carrier binding. ESI charge states and collision cross sections of the MD structures agree with experimental data. Our results confirm the Rayleigh/charged residue model (CRM). Field emission of excess Na(+) plays an ancillary role by governing the net charge of the shrinking droplet. Models that envision protein ejection from the droplet are not supported. Most nascent CRM ions retain native-like conformations. For unfolded proteins ESI likely proceeds along routes that are different from the native state mechanism explored here.

Compaction of Duplex Nucleic Acids upon Native Electrospray Mass Spectrometry

“…45 Briefly optimized structures often have CCS values matching well with the experiments. 42,[46][47][48] Fabris and co-workers have recently underlined the difficulties in transposing to DNA the MD and CCS calculation protocols traditionally used for proteins. 43 As a way out they proposed to calibrate all traveling wave IMS data using short MD simulation results, but our study shows why this approach would lead to a misrepresentation of nucleic acid structures in the gas phase.…”

“…Interestingly, the resulting charge density is similar to those observed experimentally from native conditions. It is therefore likely that the DNA duplexes are desolvated via the charged residue model 41,42 and that the compaction results from the association of NH 4 + cations to both minor and major groove before full desolvation. As a result, the starting structure for gas phase simulations might be quite different to the canonical A-or B-helices.…”

Effects of Multidentate Metal Interactions on the Structure of Collisionally Activated Proteins: Insights from Ion Mobility Spectrometry and Molecular Dynamics Simulations

“…An in-house Fortran program was used for electrostatic mapping. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 8 It is not clear from the literature 41,53 to what extent nonspecific metal adducts affect the structures of globular proteins under gentle ESI conditions. Figure 2A shows that Ω values of [Ubq + 6H] 6+ , [Ubq + 6Na] 6+ , and [Ubq + 3Ca] 6+ differ by no more than 2%.…”

“…For example, ESI of NaCl-containing protein solutions generates heterogeneous [M + zH + n(Na-H) + m(Cl+H)] z+ assemblies. [52][53][54] It has been reported that non-specifically bound small ions can stabilize collisionally activated multi-protein complexes, i.e., they shift the onset of CID to higher collision energies. 37 Anions that dissociate from the protein as protonated neutrals can lower the internal energy of the analyte by evaporative cooling.…”

Structural Effects of Solvation by 18-Crown-6 on Gaseous Peptides and TrpCage after Electrospray Ionization

“…Furthermore, the molecule must be removed from solvent and ionized (or allowed to retain a net charge) in order to be observed. Typically this is achieved with electrospray ionization (ESI); however, mechanistic details for ESI are not completely understood and it is unclear how protein structure evolves during electrospray, although recent progress has been made [24,25]. Ultimately, results have been reported where native protein structure is either retained or lost upon examination in the gas phase [26,27].…”