Native ESI-MS involves the transfer of intact proteins and biomolecular complexes from solution into the gas phase. One potential pitfall is the occurrence of pH-induced changes that can affect the ...analyte while it is still surrounded by solvent. Most native ESI-MS studies employ neutral aqueous ammonium acetate solutions. It is a widely perpetuated misconception that ammonium acetate buffers the analyte solution at neutral pH. By definition, a buffer consists of a weak acid and its conjugate weak base. The buffering range covers the weak acid pK
a
± 1 pH unit. NH
4
+
and CH
3
-COO
−
are not a conjugate acid/base pair, which means that they do not constitute a buffer at pH 7. Dissolution of ammonium acetate salt in water results in pH 7, but this pH is highly labile. Ammonium acetate does provide buffering around pH 4.75 (the pK
a
of acetic acid) and around pH 9.25 (the pK
a
of ammonium). This implies that neutral ammonium acetate solutions electrosprayed in positive ion mode will likely undergo acidification down to pH 4.75 ± 1 in the ESI plume. Ammonium acetate nonetheless remains a useful additive for native ESI-MS. It is a volatile electrolyte that can mimic the solvation properties experienced by proteins under physiological conditions. Also, a drop from pH 7 to around pH 4.75 is less dramatic than the acidification that would take place in pure water. It is hoped that the habit of referring to pH 7 solutions as ammonium acetate “buffer” will disappear from the literature. Ammonium acetate “solution” should be used instead.
Graphical Abstract
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The mechanisms whereby protein ions are released into the gas phase from charged droplets during electrospray ionization (ESI) continue to be controversial. Several pathways have been proposed. For ...native ESI the charged residue model (CRM) is favored; it entails the liberation of proteins via solvent evaporation to dryness. Unfolded proteins likely follow the chain ejection model (CEM), which involves the gradual expulsion of stretched-out chains from the droplet. According to the ion evaporation model (IEM) ions undergo electrostatically driven desorption from the droplet surface. The IEM is well supported for small precharged species such as Na+. However, it is unclear whether proteins can show IEM behavior as well. We examined this question using molecular dynamics (MD) simulations, mass spectrometry (MS), and ion mobility spectrometry (IMS) in positive ion mode. Ubiquitin was chosen as the model protein because of its structural stability which allows the protein charge in solution to be controlled via pH adjustment without changing the protein conformation. MD simulations on small ESI droplets (3 nm radius) showed CRM behavior regardless of the protein charge in solution. Surprisingly, many MD runs on larger droplets (5.5 nm radius) culminated in IEM ejection of ubiquitin, as long as the protein carried a sufficiently large positive solution charge. MD simulations predicted that nonspecific salt adducts are less prevalent for IEM-generated protein ions than for CRM products. This prediction was confirmed experimentally. Also, collision cross sections of MD structures were in good agreement with IMS data. Overall, this work reveals that the CRM, CEM, and IEM all represent viable pathways for generating gaseous protein ions during ESI. The IEM is favored for proteins that are tightly folded and highly charged in solution and for droplets in a suitable size regime.
Electrospray ionization (ESI) generates intact gas-phase ions from analytes in solution for mass spectrometric investigations. ESI can proceed via different mechanisms. Low molecular weight analytes ...follow the ion evaporation model (IEM), whereas the charged residue model (CRM) applies to large globular species. A chain ejection model (CEM) has been proposed for disordered polymers.
In addition to serving as respiratory electron shuttle, ferri-cytochrome c (cyt c) acts as a peroxidase; i.e., it catalyzes the oxidation of organic substrates by H2O2. This peroxidase function plays ...a key role during apoptosis. Typical peroxidases have a five-coordinate heme with a vacant distal coordination site that permits the iron center to interact with H2O2. In contrast, native cyt c is six-coordinate, as the distal coordination site is occupied by Met80. It thus seems counterintuitive that native cyt c would exhibit peroxidase activity. The current work scrutinizes the origin of this structure–function mismatch. Cyt c-catalyzed peroxidase reactions show an initial lag phase that is consistent with the in situ conversion of a precatalyst to an active peroxidase. Using mass spectrometry, we demonstrate the occurrence of cyt c self-oxidation in the presence of H2O2. The newly generated oxidized proteoforms are shown to possess significantly enhanced peroxidase activity. H2O2-induced modifications commence with oxidation of Tyr67, followed by permanent displacement of Met80 from the heme iron. The actual peroxidase activation step corresponds to subsequent side chain carbonylation, likely at Lys72/73. The Tyr67-oxidized/carbonylated protein has a vacant distal ligation site, and it represents the true peroxidase-active structure of cyt c. Subsequent self-oxidation eventually causes deactivation. It appears that this is the first report that identifies H2O2-induced covalent modifications as an essential component for the peroxidase activity of “native” cyt c.
The ion evaporation model (IEM) and the charged residue model (CRM) represent cornerstones of any discussion related to the mechanism of electrospray ionization (ESI). Molecular dynamics (MD) ...simulations have confirmed that small ions such as Na+ are ejected from the surface of aqueous ESI droplets (IEM), while folded proteins in native ESI are released by water evaporation to dryness (CRM). ESI of unfolded proteins yields M + zH z+ ions that are much more highly charged than their folded counterparts. A chain ejection model (CEM) has been proposed to account for the protein ESI behavior under such non-native conditions (Konermann, L., et al. Anal. Chem. 2013, 85, 2–9). The CEM envisions that unfolded proteins are driven to the droplet surface by hydrophobic and electrostatic factors, followed by gradual ejection via intermediates where droplets carry extended protein tails. Thus far, it has not been possible to support the CEM through MD simulations using realistic protein models and atomistic force fields. Such endeavors require much larger droplets than in previous MD studies. Also, the incorporation of CEM-related H+ migration is difficult. This work overcomes these challenges in MD simulations on unfolded apo-myoglobin (aMb) in droplets with a 5.5 nm radius (∼22500 water molecules). We focused on solutions at pH ∼4 where the aMb solution charge coincides with the charge on some of the electrosprayed ions (22+ to 27+), such that H+ migration could be neglected. Na+ ions were added to ensure a droplet charge close to the Rayleigh limit. We found that 16 of 17 MD runs on various protonation patterns produced M + zH z+ ions via chain ejection. The predicted stretched-out aMb conformations were consistent with experimental collision cross sections. These results support the view that unfolded proteins follow the CEM. Overall, the IEM/CRM/CEM triad can account for a wide range of ESI scenarios involving various types of analytes.
The mechanism whereby macromolecular analytes are transferred into the gas phase during the final stages of electrospray ionization (ESI) remains a matter of debate. In this work, we employ molecular ...dynamics simulations to examine the temporal behavior of nanometer-sized aqueous ESI droplets containing a polymer chain and excess ammonium ions. The polymer is modeled using a coarse-grained framework where a bead-string backbone is decorated with side chains that can be nonpolar, cationic, or anionic. Polymers that adopt compact conformations and that carry a large number of charged side chains remain close to the droplet center, where the charges are extensively hydrated. The ESI process for these compact/hydrophilic macromolecules must involve solvent evaporation to dryness. This behavior is consistent with the charged residue model (CRM). A completely different scenario is encountered for disordered (extended) chains that carry a large number of nonpolar side chains. In this case, the macromolecule tends to be rapidly expelled from the droplet surface in a stepwise sequential fashion. This process produces metastable structures where one end of the extended polymer chain remains connected with the droplet via charge solvation. Disruption of these last interactions will then produce a free gas phase macromolecular ion. The data of this work imply that the ESI process for unfolded/hydrophobic polymers proceeds via an ion evaporation (IEM)-like mechanism that is facilitated by hydrophobic solute/solvent interactions. Our model predicts that the ESI efficiency of the latter scenario is considerably higher than for the CRM. This prediction is verified experimentally through ESI mass spectrometry measurements on myoglobin.
The ejection of solvated small ions from nanometer-sized droplets plays a central role during electrospray ionization (ESI). Molecular dynamics (MD) simulations can provide insights into the ...nanodroplet behavior. Earlier MD studies have largely focused on aqueous systems, whereas most practical ESI applications involve the use of organic cosolvents. We conduct simulations on mixed water/methanol droplets that carry excess NH4 + ions. Methanol is found to compromise the H-bonding network, resulting in greatly increased rates of ion ejection and solvent evaporation. Considerable differences in the water and methanol escape rates cause time-dependent changes in droplet composition. Segregation occurs at low methanol concentration, such that layered droplets with a methanol-enriched periphery are formed. This phenomenon will enhance the partitioning of analyte molecules, with possible implications for their ESI efficiencies. Solvated ions are ejected from the tip of surface protrusions. Solvent bridging prior to ion secession is more extensive for methanol/water droplets than for purely aqueous systems. The ejection of solvated NH4 + is visualized as diffusion-mediated escape from a metastable basin. The process involves thermally activated crossing of a ∼30 kJ mol–1 free energy barrier, in close agreement with the predictions of the classical ion evaporation model.
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.
The structure and reactivity of electrosprayed protein ions is governed by their net charge. Native proteins in non-denaturing aqueous solutions produce low charge states. More highly charged ions ...are formed when electrospraying proteins that are unfolded and/or exposed to organic supercharging agents. Numerous studies have explored the electrospray process under these various conditions. One phenomenon that has received surprisingly little attention is the charge enhancement caused by multivalent metal ions such as La3+ when electrospraying proteins out of non-denaturing solutions. Here, we conducted mass spectrometry and ion mobility spectrometry experiments, in combination with molecular dynamics (MD) simulations, to uncover the mechanistic basis of this charge enhancement. MD simulations of aqueous ESI droplets reproduced the experimental observation that La3+ boosts protein charge states relative to monovalent metals (e.g., Na+). The simulations showed that gaseous proteins were released by solvent evaporation to dryness, consistent with the charged residue model. Metal ion ejection kept the shrinking droplets close to the Rayleigh limit until ∼99% of the solvent had left. For droplets charged with Na+, metal adduction during the final stage of solvent evaporation produced low protein charge states. Droplets containing La3+ showed a very different behavior. The trivalent nature of La3+ favored adduction to the protein at a very early stage, when most of the solvent had not evaporated yet. This irreversible binding via multidentate contacts suppressed La3+ ejection from the vanishing droplets, such that the resulting gaseous proteins carried significantly more charge. Our results illustrate that MD simulations are suitable for uncovering intricate aspects of electrospray mechanisms, paving the way toward an atomistic understanding of mass spectrometry based analytical workflows.
