Daniel A. Abaye*a,
Irene A. Agboab and
Birthe V. Nielsenc
aDepartment of Basic Sciences, School of Basic and Biomedical Sciences, University of Health and Allied Sciences, PMB 31, Ho, VR, Ghana. E-mail: dabaye@uhas.edu.gh
bNelson Mandela University, Department of Chemistry, Port Elizabeth, South Africa
cSchool of Science, Faculty of Engineering and Science, University of Greenwich, Chatham Maritime, Kent, ME4 4TB, UK
First published on 7th June 2021
In electrospray ionization mass spectrometry (ESI-MS), analytes are introduced into the mass spectrometer in typically aqueous-organic solvent mixtures, including pH modifiers. One mechanism for improving the signal intensity and simultaneously increasing the generation of higher charge-state ions is the inclusion of small amounts (approx. <0.5% v/v mobile phase solution) of charge-inducing or supercharging reagents, such as m-nitrobenzyl alcohol, o-nitrobenzyl alcohol, m-nitrobenzonitrile, m-(trifluoromethyl)-benzyl alcohol and sulfolane. We explore the direct and indirect (colligative properties) that have been proposed as responsible for their modes of action during ESI. Of the many theorized mechanisms of ESI, we re-visit the three most popular and highlight how they are impacted by supercharging observations on small ions to large molecules including proteins. We then provide a comprehensive list of 34 supercharging reagents that have been demonstrated in ESI experiments. We include an additional 19 potential candidate isomers as supercharging reagents and comment on their broad physico-chemical properties. It is becoming increasingly obvious that advances in technology and improved ion source design, analyzers e.g. the use of ion mobility, ion trap, circular dichroism (CD) spectroscopy, together with computer modeling are increasing the knowledge base and, together with the untested isomers and yet-to-be unearthed ones, offer opportunities for further research and application in other areas of polymer research.
In this brief review, we provide a comprehensive list of 34 supercharging reagents that have been used in studies and include an additional 19 potential candidate isomers commenting on their broad physico-chemical properties. These suggested isomers have the potential to further enhance our understanding of supercharging during ESI-MS. This report may be viewed as a first compendium or a ‘one-stop-shop’ of supercharging reagents for fellow ESI mass spectrometrists to peruse.
In ESI, one of the main approaches for increasing the generation and detection of higher charge state in peptide ions (i.e. from [M + nH]n+ to [M + (n + 1)H]n+1)+ and simultaneously improving the signal intensity (ESI response) is the inclusion of very small amounts (approx. <5% v/v of mobile phase solution) charge-inducing compounds or supercharging reagents. Supercharging reagents include glycerol,5 m-nitrobenzyl alcohol (mNBA; 3-nitro(phenyl) methanol),4,6,7 o-nitrobenzyl alcohol (2-nitro(phenyl)methanol) 8 and sulfolane.8,9 Thus far, the supercharging reagents used in experiments are all small molecules (mol. wt. <180 Da (Table 1).
Common name/[IUPAC name]α | Molecular structureα | Ave. mass,α Da | Boiling ptα °C at 760 mmHg | Vapour press.αβ mmHg at 25 °C | Surface tensionαβ mN m−1 at 25 °C | Densityαβ (specific gravity) 25 °C | Acidity pKa 25 °C | Dipole momentδ μ(D)/μD* | Ref. | |
---|---|---|---|---|---|---|---|---|---|---|
a Key: α chemspider: http://www.chemspider.com/, γ pKa ε candidate isomers proposed supercharging reagents, β pubchem: https://pubchem.ncbi.nlm.nih.gov/, δ dipole moment μ(D)/μD*: determined by experiment/calculated*. We acknowledge that some values in Table 1 are incomplete. | ||||||||||
3- to 4-membered & planar molecules | ||||||||||
1 | Dimethyl sulfoxide (DMSO) [(Methylsulfinyl)methane] | 78.133 | 189 | 0.60 | 43.54 | 1.101 (ref. 65) | 35.1 | 3.96/4.44 | 3 and 25 | |
2 | Ethylene glycol [1,2-ethandiol] | 62.068 | 196–198 | 0.06 | 47.99 | 1.113 | 14.22 | 2.747/— | 3 | |
3 | Glycerol [1,2,3-propanetriol] | 92.094 | 182 | 1.68 × 10−4 | 72.6 | 1.26 (ref. 65) | 14.4 | 2.68/— | 5 and 8 | |
4 | Formamide | 45.041 | 210 | 6.10 × 10−2 (ref. 66) | 58.35 (ref. 67) | 1.13 | 23.5 (in DMSO) | — | 3 | |
5 | 2-Methoxyethanol | 76.094 | 124–125 | 6.2 (ref. 68) | 30.84 (ref. 69) | 0.97 (ref. 65 and 70) | 14.8 | — | 3 | |
6ε | Methoxypropanol [1-methoxypropan-2-ol] | 90.122 | 120 | 10.9 | 2.64 × 10−3 | 0.926 | 14.49 ± 0.20 (ref. 70 and 71) | — | ||
7 | Crotononitrile[(2E)-2-butenenitrile] | 67.089 | 120–121 | 31.95 | — | — | — | 4.3/— | 15 | |
Heterocyclic 4- & 5-membered ring structures, including organosulfur cpd (sulfones) | ||||||||||
8 | Dimethyl sulfone [methylsulfonylmethane] | 94.133 | 238 | — | — | 1.45 | 31 | 4.5/— | 15 | |
9 | N,N,N′N′-Tetraethylsulfamide (TES) [N-(diethylsulfamoyl)-N-ethylethanamine] | 208.32 | 125 | — | — | 1.283 | — | — | 16 | |
10 | 3-Chlorothiete-1,1-dioxide | 140.59 | 328.9 | — | — | 1.64 | — | —/3.38 | 9 | |
11ε | 3-Chloro-2H-thiete 1,1-dioxide | 138.573 | 328.9 ± 41.0 | 3.2 ± 0.3 | — | 1.6 ± 0.1 | — | — | ||
12 | 2-Thiophenone [2H-thiophen-5-one] | 100.139 | 197.4 | 0.4 ± 0.3 | 38.7 ± 3.0 | 1.24 | 49 | |||
13ε | 4-Butyrothiolactone [thiolan-2-one] | 102.16 | 76.5429 | — | — | 1.18 | Poorly soluble in water | — | ||
14 | Sulfolane [2,3,4,5-tetrahydrothiophene-1,1-dioxide] | 120.170 | 285 | 6.2 × 10−3 (27.6 °C) | 35.5 (ref. 72) | 1.26 (ref. 65) | 12.9 | 4.35/5.68 | 7, 8, 52 and 53 | |
15 | Sulfolene [2,5-dihydrothiophene 1,1-dioxide] | 118.154 | 64–65.5 °C mpt (ref. 73) | — | 41.0 | 1.3 (ref. 68) | — | —/5.69 | 47 | |
16 | 1,3-Propanesultone [1,2-oxathiolane 2,2-dioxide] | 122.143 | 315.87 | — | — | 1.392 | — | — | 15 | |
17 | 1,4-Butanesultone [1,2-oxathiane 2,2-dioxide] | 136.169 | 304.047 | — | — | 1.335 | — | — | 15 | |
18 | 3-Methyl-2-oxazolidone (MOZ) [3-methyl-1,3-oxazolidin-2-one] | 101.1 | 88 | — | — | 1.17 | — | — | 16 | |
Heterocyclic 4- & 5-membered ring structures (including the heterocyclic acetals) | ||||||||||
19 | Glycerol carbonate [4-hydroxymethyl-1,3-dioxolan-2-one] | 118.088 | 353.9 ± 15 | 0.0 ± 1.8 | 41.1 | 1.375 | — | — | 48 | |
20 | Propylene carbonate [4-methyl-1,3-dioxol-2-one] | 102.089 | 240 | 0.045 (ref. 66) | 45 | 1.2 (ref. 69) | — | 4.9 | 48 and 49 | |
21 | Ethylene carbonate [1,3-dioxolan-2-one] | 88.062 | 244–245 | — | 54 (30 °C) | 1.32 | — | 4.9/— | 15 | |
22 | Butylene carbonate [4-ethyl-1,3-dioxolan-2-one] | 116.115 | 281.18 | — | — | 1.141 | — | — | 15 | |
23 | Vinylethylene carbonate [4-vinyl-1,3-dioxolan-2-one] | 114.099 | 238.72 | — | — | 1.188 | — | — | 15 | |
Benzene-ring containing molecules | ||||||||||
24 | Nitrobenzene | 123.109 | 210–211 | 4.2 | 0.245 | 1.201 | 1.204 slightly soluble in water | 4.2/— | 15 | |
25 | Benzyl alcohol [(hydroxymethyl)benzene] | 108.138 | 205 | 0.094 (ref. 66) | 39.0 (ref. 70) | 1.0 (ref. 65) | 15.4 | 1.71/1.79 | 8 | |
26 | m-Chlorophenol [3-chlorophenol] | 128.556 | 214 | 0.125 | 44.7 | 1.3 (ref. 68) | 9.12 | 1.03 ± 1.08 | 4 | |
27ε | o-Chlorophenol [2-chlorophenol] | 128.556 | 175–176 | 2.53 (ref. 66) | 40.50 | 1.26 (ref. 65) | 8.56 | — | ||
28ε | p-Chlorophenol [4-chlorophenol] | 128.556 | 217–220 | 1 (ref. 74) | 8.7 × 10−2 mm Hg | 1.306 (ref. 65) | 9.41 | 2.10/— | ||
29 | 2-Nitrochlorobenzene [1-chloro-2-nitrobenzene] | 157.555 | 245–246 | — | 48.4 ± 3.0 | 1.348 | 0.6 | 4.6/— | 15 | |
30ε | 3-Nitrochlorobenzene [1-chloro-3-nitrobenzene] | 157.555 | 235–236 | 0.097 (ref. 66) | 4.37 × 10−2 | 1.3 | — | — | ||
31ε | 4-Nitrochlorobenzene [1-chloro-4-nitrobenzene] | 157.555 | 241.66 | 2.19 × 10−2 (ref. 66) | 3.71 × 10−2 | 1.520 | n/a | — | ||
32 | o-Nitrobenzyl alcohol (oNBA) [2-nitro(phenyl)methanol] | 153.135 | 270 (solid at RT) | 0.0 ± 0.5 | — | 1.3 | — | — | 8 | |
33 | m-Nitrobenzyl alcohol (mNBA) [3-nitro(phenyl) methanol] | 153.135 | 349.8 | 0.0 ± 0.5 | 50 ± 5 | 1.29 | — | — | 4, 6, 7, 8, 48, 49 and 75 | |
34 | p-Nitrobenzyl alcohol (pNBA) [4-nitro(phenyl)methanol] | 153.135 | 185 (solid at RT) | Negligible | — | — | 7.15 | — | 8 | |
35 | m-Nitroacetophenone [1-(3-nitrophenyl)ethanone] | 165.146 | 202 (solid at RT) | 3.86 × 10−5 | — | 1.4 | — | — | 8 and 27 | |
36 | m-Nitrobenzonitrile [3-benzonitronitrile] | 148.119 | 165 | 0.0 ± 0.5 | — | 1.3 ± 0.1 | — | — | 6, 8 and 27 | |
37ε | o-Nitrobenzonitrile [2-benzonitronitrile] | 148.119 | 321.78 165.0 °C (16.0 mmHg) | Insoluble in water | — | — | — | — | ||
38ε | p-Nitrobenzonitrile [4-benzonitronitrile] | 148.119 | — | — | — | — | — | — | ||
39 | m-nitrophenyl ethanol [1-(3-nitrophenyl)ethanol] | 167.162 | 281.3 ± 23.0 | 0.0 ± 0.6 | — | 1.3 ± 0.1 | — | — | 6 and 8 | |
40ε | o-Nitrophenyl ethanol [1-(2-nitrophenyl)ethanol) | 167.162 | 319.0 ± 17.0 | 0.0 ± 0.6 | — | 1.3 ± 0.1 | — | — | ||
41ε | p-Nitrophenyl ethanol (1-(4-nitrophenyl)ethanol] | 167.162 | 290.68 | 0.0 ± 0.7 g | — | 1.3 ± 0.1 | — | — | ||
42 | o-Nitroanisole [1-methoxy-2-nitrobenzene] | 153.135 | 272–273 | — | 48 | 1.254 | — | 5.0/— | 15 | |
43ε | m-Nitroanisole [1-methoxy-3-nitrobenzene] | 153.135 | 256.3 ± 13.0 | 0.0 ± 0.5 | — | 1.2 ± 0.1 | — | — | ||
44 | p-Nitroanisole [1-methoxy-4-nitrobenzene] | 153.135 | 260 | — | — | 1.233 | — | 5.3/— | 15 | |
45 | 3-Nitrophenethyl alcohol [2-(3-nitrophenyl)ethanol] | 167.162 | 341.7–349.0 | — | — | — | — | — | 8 | |
46ε | 2-Nitrophenethyl alcohol [2-(2-nitrophenyl)ethanol] | 167.162 | 267 | — | — | 1.19 | — | — | ||
47ε | 4-Nitrophenethyl alcohol [2-(4-nitrophenyl)ethanol] | 167.162 | 337 | — | — | — | — | — | ||
48 | m-(Trifluoromethyl)-benzyl alcohol [[3-(trifluoromethyl)phenylmethanol] | 176.136 | 257–261 | 0.1 ± 0.4 | — | 1.3 ± 0.1 | — | — | 6 | |
49ε | o-(Trifluoromethyl)-benzyl alcohol [2-(trifluoromethyl)phenylmethanol] | 176.136 | 214–262 | — | — | 1.3 ± 0.1 | — | — | ||
50ε | p-(Trifluoromethyl)-benzyl alcohol [4-(trifluoromethyl)benzyl alcohol] | 176.136 | 250 | 0.1 ± 0.4 | — | 1.28 (ref. 74) | — | — | ||
51ε | o-Nitrobenzoic acid [2-nitrobenzoic acid] | 167.12 | 295.67 | 0.0 ± 0.8 (ref. 75) | 66.4 ± 3.0 | 1.575 | 2.17 | 4.07/— | ||
52ε | m-Nitrobenzoic acid [3-nitrobenzoinc acid] | 167.12 | 340.7 ± 25.0 | 3.71 × 10−5 (ref. 75) | 66.4 ± 3.0 | 1.5 ± 0.1 | 3.47 | 4.03/— | ||
53ε | p-Nitrobenzoic acid [4-nitrobenzoic acid] | 167.12 | It sublimes | — | — | 1.58 | 3.41 | 4.05/— | ||
Typical solvents used in ESI | ||||||||||
Common name/IUPAC name | Molecular structure | Ave. mass, Da | Boiling pt °C at 760 mmHg | Vap. press. mmHg at 25 °C (ref. 80) | Surface tension mN m−1 at 25 °C | Density (specific gravity) | Acididty pKa 25 °C | Dipole moment μ(D)/μD* | 66 and 69 | |
1 | Water | 18.02 | 100 | 17.44 | 72.8 | 1.00 | 14 | 1.85/2.16 | ||
2 | Methanol | 32.04 | 64.7 | 127 | 22.07 | 0.792 | 15.5 | 1.70/ | ||
3 | Ethanol | 46.07 | 78.2 | 59.3 | 21.97 | 0.789 | 15.9 | 1.69/ | ||
4 | Acetonitrile | 41.05 | 81.3–82.1 | 88.8 | 29.04 | 0.786 | 25 | 3.92/ | ||
5 | Ethyl acetate | 88.106 | 77.1 | 93.2 | 24 | 0.902 | 25 | 1.78/ | ||
6 | Acetone | 58.08 | 56.05 | 231 | 23.7 | 0.785 | 19.16 | 2.91/ |
(1) Their ability to raise the solution surface tension.10 Due to the low volatility of these compounds compared with that of common mobile-phase compositions (e.g. water/methanol or water/acetonitrile), their concentration increases as evaporation of the more volatile components from the droplets occurs. This enrichment of the supercharging reagents raises the surface tension which results in a requirement for a higher degree of surface charging to reach the Rayleigh limit for a spherical droplet. This, in turn, increases the boiling point of the supercharging reagent-enriched environment10 However, Samalikova and Grandori11 reported that their results were not easily reconciled by considering only charge availability of the precursor droplets at the Rayleigh limit as determined by surface tension.
(2) Protein denaturation: Williams and co-workers argued that protein unfolding induced by chemical and or thermal denaturation appears to be the primary origin of the enhanced charging observed in the protein complexes investigated.7 In their experiments, the arrival time distributions obtained from traveling wave ion mobility spectrometry showed that the higher charge state ions that are formed with m-NBA and sulfolane are significantly more unfolded than lower charge state ions. Sterling et al.12 argued that droplet heating, owing to the high boiling point of m-NBA, resulted in thermal denaturation. However, unlike Sterling et al.12 Hogan et al.13 reported that supercharging reagents do not cause structural protein modifications in solution and that the modest mobility decrease observed could be partly attributed to trapping of the sulfolane within the protein ions.
Further, the study by Douglass and Venter demonstrated that supercharging was shown to increase with increasing dipole moment for the supercharging reagents sulfolane and sulfolene.9 Finally, if an increase in surface tension is the cause of supercharging, then it should occur in both the positive and negative modes since the effect of surface tension on the Rayleigh limit is independent of the polarity of the charge. Like many other identified supercharging reagents, sulfolane is highly polar with a dipole moment of 4.35 D, much higher than that of water (1.85 D) or methanol (1.70 D) (Table 1).
The interaction of supercharging reagents with charged basic sites would depend on the molecular structure, such as the type and location of functional groups. Thus, they exhibit low solution-phase basicities and relatively low gas-phase basicities.8 For example, the most effective supercharging reagents have one or more carbonyl, sulfonyl, or nitro groups present in their structure (Table 1), which may be important for intermolecular interactions between the supercharging reagent and the charged basic site. Lomeli et al. demonstrated that supercharging occurs not only when liquid m-NBA is present, but also with solid o- or p-isomers. This provides some evidence against increased surface tension causing supercharging.8
A report evaluating the potential of low volatility of supercharging reagents demonstrated that there was not a strong correlation between the extent of analyte (protein) charging with either surface tension, dipole moment, or dielectric constant of the additives.15 That is, there was no single physical property of an additive that significantly determined the extent of protein charging when formed from a solution mixture. What is clear, however, was that most of the supercharging reagents have higher boiling points, higher surface tension values, high dielectric constants, and higher dipole moments than the ESI solvents. Thus, a combination of these physicochemical properties together with other factors including adduct formation between analyte and supercharger, basicity at the solution, and gaseous phases, may all influence charging, although the extent of charging may depend on the experimental conditions.
A comprehensive list of 53 molecules of which 34 have been shown to have supercharging ability in experiments and 19 potential candidate isomers, together with some of their physico-chemical properties are given in Table 1. The molecules are grouped conveniently in terms of molecular structure and functional group given rise to four categories:
(1) Acyclic 3- to 4-membered and/or planar structures.
(2) Heterocyclic 4- to 5-membered molecules including the sulfones and S-containing ones. Dimethyl sulfone and N,N,N′N′-tetraethylsulfamide (TES) are acyclic molecules but are included in this category because they contain the sulfone functional group.
(3) Heterocyclic 4- to 5-membered molecules including the heterocyclic acetals.
(4) Molecules containing the benzene structure with at least two functional group attachments which may have opposite charges e.g. m-nitrobenzyl alcohol.
Recently, two new supercharging reagents N,N,N′,N′-tetraethylsulfamide (TES), and 3-methyl-2-oxazolidone (MOZ) were shown to counter the suppression activity of trifluoroacetic acid (TFA).16 The study also demonstrated that the two molecules increased charge states of peptides and proteins and improved separation efficiency during reverse-phase LC-MS determinations.
We suggest that the following 19 additives (denoted by ε and in italics, Table 1) are potential supercharging reagents and could be evaluated:
(1) Based on the fact that some of their isomers have successfully been demonstrated; methoxypropanol, 3-chloro-2H-thiete 1,1-dioxide, 4-butyrothiolactone, o-chlorophenol, p-chlorophenol, 3-nitrochlorobenzene, 4-nitrochlorobenzene, o-nitrophenylethanol, p-nitrophenyl ethanol, m-nitroanisole, o-(trifluoromethyl)-benzyl alcohol, and p-(trifluoromethyl)-benzyl alcohol, and,
(2) Molecules having a benzene ring structure and with two or more attached functional groups having opposing charges (‘dual polarity’ isomers); e.g. the isomers o-, m-, and p- of nitrobenzoic acid, nitrobenzonitrile and nitrophenethyl alcohol.
Fig. 1 A simple illustration of the ESI environment. For simplicity, droplets are presented with positive charges. According to the model presented by Loo et al.27 opposing charges may also be present in the gas phase ion. Under an electric field, elevated temperature but at ambient pressure, the sprayed analyte solution undergoes the simultaneous processes of the droplet splitting, generation of highly charged droplets, repeated splitting and shrinkage of the droplets, solvent evaporation, and removal under a directed gas (typically N2) stream. These progeny droplets themselves undergo subsequent and repeated evaporation and splitting, and this process is repeated until only gas-phase ions are generated.76 The ions are then drawn under negative pressure into the mass spectrometer. |
Observations show that many parameters e.g. solvent composition,3,4,17,18 analyte composition and concentration,19–21 pH, flow rate,18,21 denaturing solutions,22,23 and non-denaturing solutions,24–26 solution- & gas-phase basicity, solution-phase conformation,26,27 instrument settings: source voltage,28,29 sprayer orifice diameter,30 gas pressures,31 and, ion source type (laser ESI-LEMS vrs conventional ESI)32 affect charge state distributions (CSD) especially, in large analytes such as proteins. Further, sub-ambient pressure ESI source combined with nano-flow significantly improves ion yield and sensitivity33
Several models have been put forward to explain the mechanisms of action of the processes of generating gas-phase ions from the liquid mobile phase. Among these, three are popular: the charge residue model (CRM),34,35 the ion evaporation model (IEM)36–40 and the third postulate, chain ejection model (CEM)41,42 which was recently described for unfolded proteins.2,43,44 These models are summarized in Table 2.
The three popular models that seek to explain the ESI mechanism | |||
---|---|---|---|
Charge residue model (CRM) | Ion evaporation model (IEM) | Chain ejection model (CEM) | |
Background | The CRM was proposed based on the ability of the SCRs to raise the surface tension of the mobile phase solvent, a requirement for a higher degree of surface charging to reach the Rayleigh limit which, results in a coulombic fission event | Following the repeated splitting and shrinkage of droplets, the size of droplets reach a certain critical radius (10−6 cm = 10−8 m), the field strength at the surface of the droplet becomes large enough to assist thefield desorptionof solvated ions | Neither the CRM nor IEM mechanisms explain quantitatively the extent of macromolecular multiple charging, especially in unfolded proteins |
Key points in the mechanism | Millions of small, highly charged progeny droplets containing the analyte leave the parent droplet with a disproportionately large fraction of the surface charge | Analyte ions desorb directly from charged nanodroplets, driven by the large electric field at the droplet surface | The inclusion of SCRs has opened up new ways to modulate protein charge states, thereby challenging the existing ESI models |
Progeny droplets containing single analytes are charged through a charge transfer process between the charge carriers on the surface of the droplet and the analyte molecule upon final droplet evaporation | The ion evaporation process then becomes operative for a highly electrified cloud of droplets at low solute concentrations | ESI of unfolded proteins yields [M + (z + 1)H]z+ ions that are much more highly charged than their folded counterparts | |
That is, analyte ions desorb directly from charged nanodroplets, driven by the large electric field at the droplet surface | Solvent properties such as dipole moment and protein–SCR adduct formation have been shown to play prominent roles in supercharging in proteins | ||
Analytes | Larger ions (e.g. from folded proteins under non-denaturing conditions) are formed by CRM | It has been demonstrated that small ions (i.e. Na+), generated from small molecules, are liberated into the gas phase through the ion evaporation mechanism | This proposed model accounts for the protein ESI behavior under such non-native conditions and recently has been proposed to apply to unfolded proteins |
Illustration | |||
References | 34, 77 and 78 | 10, 36–40 and 79 | 2, 41–44, 52 and 53 |
The use of nano-ESI, conventionally a non-pneumatic operation with very low mobile flow rates (∼<1 μL min−1) and much small samples sizes which then provides improved desolvation, greater salt tolerance, and a higher ion yield29,33 has added another dimension to the debate although the generated final ions are believed to be the same for both conventional and nano-ESI.45
Hogan et al. proposed that both the IEM and CRM are in play, in which macromolecules are charged residues but, in negative ion mode, carry less charge because highly-charged droplets field-emit anions. Why positively charged droplets would not also field-emit small ions has not yet been explained.46,47 Excellent discussions are also given.9,27,48,49
Proteins that are unfolded in solution produce higher charge states during ESI than their natively folded counterparts. Building on the work by Douglass and Venter,9 who showed experimentally the formation of adducts between sulfolane and the most highly charged protein ions, Peters et al. recently, reported that the CEM involves the partitioning of mobile H+ (e.g. from formic acid) between the droplet and the departing protein.44 Their results indicate that the supercharging of unfolded proteins is caused by residual sulfolane that stabilizes protonated sites on the protruding chains. This, therefore, promotes H+ retention on the protein. Their report suggested that charge stabilization on the sites of projecting chains is due to charge–dipole interactions which are mediated by the large dipole moment and the low volatility of sulfolane.
Denaturing ESI follows the CEM, where protein ions are gradually expelled from the droplet surface. Proton (H+) equilibration between the droplets and the protruding chains culminates in highly charged gaseous proteins.2 The presence of internal disulfide (S–S) bonds on the extent of supercharging was determined in three proteins each containing multiple internal disulfide bonds; bovine serum albumin, β-lactoglobulin, and lysozyme.43 Reduction of the disulfide bonds led to a marked increase in charge state following the addition of sulfolane without significantly altering folding in solution. This evidence supports a supercharging mechanism in which these proteins unfold before or during evaporation of the electrospray droplet and ionization would therefore, occur by the CEM.
Ogorzalek Loo et al. proposed a three-regime view of ESI, i.e. with solution, intermediate, and gas-phase regimes in the ESI environment.27 The intermediate phase was introduced to explain the charge transfer and other accommodations that analytes especially protein ions undergo when exiting the bulk solution, this concept rationalizes observations that ion intensities vary little over pH 3–11, despite huge changes in solution compositions.17,54,55 This kinetic model is unique in treating the evaporating droplets as charge-polarized and uses their decompositions and the variation in solution-to-gas phase properties to explain charge enrichment in ESI.
The 3-phase ESI environment could, in principle, be imagined for the IEM, CRM, and other models, particularly as distinctions between them blur in attempts to explain non-conforming observations.29,56 Thus far, the IEM and the CRM appear to represent the foundations of any discussion related to the mechanism of ESI. Molecular dynamics (MD) simulations52,57 suggest that small ions such as Na+ are ejected from the surface of an aqueous ESI droplet (IEM), while folded proteins in native ESI could be released by water evaporation to dryness (CRM).43,52,53,58
Other factors that increase the charge state during ESI are the diameter of the emitter opening: proteins (native and denatured) charge state distributions (CSDs) undergo a small, but reproducible shift to higher charge when delivered by nano-spray versus standard electrospray59,60 and source voltage.26,61 Ogorzalek Loo, et al.27 noted that this relationship between the extent of charging and initial droplet diameter could not be observed for ions released as charge residues, thus, these observations argue against CRM being the primary source of native-like ions.
Improvements in ESI source design including sub-ambient pressure with nano-flow,33,62,63 chemically-etched emitters with sheath gas capillaries and nano-flow64 are reported to enhance signal intensity, increase desolvated ion transmission to the mass spectrometer proper and thus increasing overall sensitivity. The use of these improvements together with supercharging reagents should further enhance our understanding of the ESI mechanism and how it is impacted by the supercharging reagents.
(2) The application of ESI-MS in protein analysis and the inclusion of supercharging reagents (SCR) has been the main driver in the exploration of the mechanism of the ESI process and the dynamics governing proteins in the native and unfolded state.
(3) The Konermann team2,44,52,53 and Donor et al.43 indicated that the CEM largely explains the CSD in proteins as it accounts for the dipole-moment of solvents, charge distribution from the folded to the unfolded state in proteins, and also the presence of intramolecular di-sulfide bonds within proteins.
(4) SCRs have expanded and increased our knowledge of the ESI environment and protein unfolding. These molecules have boiling points and higher surface tension than the solvent mixtures used in ESI,10 although studies by Samalikova and Grandori11 reported a diminished role of surface tension. The vast majority of supercharging molecules have densities greater than that of water and other ESI solvents (Table 1). William's and Loo's teams observed that SCRs also have low solution-phase basicities (Brønsted bases weaker than H2O) and relatively low gas-phase basicities.26–28 They also have higher dipole moments (except for acetonitrile (3.92 D)). In solution, SCRs are ionized, and highly polarized.
(5) Loo's team8 suggested that for dual-polarity superchargers, the molecule should both be a weak Brønsted base and a weak Brønsted acid.
(6) Venter's team9 demonstrated supercharging in cytochrome in the positive ionization mode, however, no change in the CSD was observed in the negative mode. This, therefore, diminishes the role of polarity-independent factors such as conformational changes or surface tension effects as key vehicles for supercharging.
(7) Venter's team9 also demonstrated that when a SCR is added in concentration about equal to or greater than the proteins concentration in solution, during ESI, there is adduct formation between the proteins and SCR.
(8) Using SCRs such as nitrobenzyl alcohol, where there are two functional groups (hydroxyl and nitro), supercharging increases in the order of the para, meta, and ortho isomers. The same principle could perhaps be applied to the isomers of nitrochlorobenzene, nitroanisole, nitrobenzoic acid, nitrobenzonitrile, and nitrophenethyl alcohol.
(9) Improvements in ion source design to include operating at sub-ambient pressure combined with nano-flow increases desolvation of charged species and elevates the ion transmission rate into the analyser section.33,62–64
It should be pointed out that all these different supercharging attributes have been proposed to matter, but not all are of them are relevant. Experimental conditions would increase the prominence of one or several of them over the others depending on the aims of the researchers.
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