Effects of liquid phase composition on salt cluster formation in positive ion mode electrospray mass spectrometry: implications for clustering mechanism in electrospray

Potassium bromate salt clusters, [KBrO3]nKx(x+), formed by electrospray ionization were studied as a function of solution properties. Clusters with up to 4 positive charges were observed. Their abundance, charge state and distribution were shown to vary with the organic solvent in solution. The effects of 7 solvents, including methanol, ethanol, isopropanol, acetonitrile, acetone, pyridine, and 1,4-dioxane, were thoroughly investigated. Solvents with a low dielectric constant and a high viscosity seem to favor clustering in solution but do not systematically allow high charge state ion formation. On the other hand, cluster charge reduction during desolvation was not correlated with solvent cation affinity over the range of solvents examined. However, ion distribution in mass spectra could be rationalized as a combination of these two competing phenomena. Charge state increases with the cluster size but may be reduced during ion desolvation when high cation affinity solvent molecules are actually involved in the ion solvation shell. This assumption could be envisaged in either Iribarne or Dole mechanisms of ion release in the gas phase. However, intensity profiles of multiply charged clusters could only be understood in terms of the ion evaporation mechanism.     

Charge state separation for protein applications using a quadrupole time-of-flight mass spectrometer

A novel method for separating ions according to their charge state using a quadrupole time-of-flight mass spectrometer is presented. The benefits of charge state separation are particularly apparent in protein identification applications at low femtomole concentration levels, where in conventional TOF MS spectra peptide ions are often lost in a sea of chemical noise. When doubly and triply charged tryptic peptide ions need to be filtered from singly charged background ions, the latter are suppressed by two to three orders of magnitude, while from 10-50% of multiply charged ions remain. The suppression of chemical noise reduces the need for chromatography and can make this experimental approach the electrospray equivalent of conventional MALDI peptide maps. If unambiguous identification cannot be achieved, MS/MS experiments are performed on the precursor ions identified through charge separation, while the previously described Q2-trapping duty cycle enhancement is tuned for approximately 1.4 of the precursor m/z to enhance intensities of ions with m/z values above that of the precursor. The resulting product ion spectra contain few fragments of impurities and provide quick and unambiguous identification through database search. The multiple charge separation technique requires minimal tuning and may become a useful tool for analysis of complex mixtures.     

Solvent effect on analyte charge state,signal intensity,and stability in negative ion electrospray mass spectrometry; implications for the mechanism of negative ion formation

Department of Chemistry, University of New Orleans, New Orleans, Louisiana, USA The effect of solvent composition on negative ion electrospray ionization (ESI) mass spectrometry was examined. The onset potentials for ES1 of a series of chlorinated solvents and methanol were found to be within the range predicted by D. P. H. Smith, based on differences in the surface tension of the solvents used. The tendency toward electric discharge decreased with increasing percent weight of chlorine in the solvent. This effect has been attributed to an increasing propensity for electron capture for more highly chlorinated solvents. Addition of the electron scavenger gas SF, was even more effective at suppressing corona discharge phenomena. In a comparison of ultimate signal intensity obtainable for a test analyte in 10% methanol, the highest signal, which was stable over the widest range of temperatures, was exhibited by chloroform compared to dichloromethane, 1,2-dichloroethane, carbon tetrachloride, and methanol (100%). Chloroform, thus, is a recommended solvent for negative ion electrospray mass spectrometry (ES/MS) when solubility is not a limiting issue. Solvent polarity was shown to exhibit a profound influence on the distribution of charge states in negative ion ES/MS. For both chlorinated and nonchlorinated organic solvents, the higher the solution dielectric constant, the more the charge-state distribution is shifted toward higher charge states. These observations build on the “electrophoretic” mechanism of droplet charging. Solvents with high solution dielectric constants are considered to be most effective at stabilizing multiply charged ions (where charge separation is greatest), and they are likely to increase the level of droplet charging. Solvents with high basicities (gas phase and solution phase) and high proton affinities, yet low dielectric constants, favor lower charge states in ES mass spectra of lipid A and cardiolipin from Escherichia coli. This indicates that gas-phase processes and solvent basicity contribute much less toward ion formation than solution-phase solvation via preferred orientation of the solvent dipole.     

Gas-phase ion/ion reactions of multiply protonated polypeptides with metal containing anions

Gas-phase reactions of multiply protonated polypeptides and metal containing anions represent a new methodology for manipulating the cationizing agent composition of polypeptides. This approach affords greater flexibility in forming metal containing ions than commonly used methods, such as electrospray ionization of a metal salt/peptide mixture and matrix-assisted laser desorption. Here, the effects of properties of the polypeptide and anionic reactant on the nature of the reaction products are investigated. For a given metal, the identity of the ligand in the metal containing anion is the dominant factor in determining product distributions. For a given polypeptide ion, the difference between the metal ion affinity and the proton affinity of the negatively charged ligand in the anionic reactant is of predictive value in anticipating the relative contributions of proton transfer and metal ion transfer. Furthermore, the binding strength of the ligand anion to charge sites in the polypeptide correlates with the extent of observed cluster ion formation. Polypeptide composition, sequence, and charge state can also play a notable role in determining the distribution of products. In addition to their usefulness in gas-phase ion synthesis strategies, the reactions of protonated polypeptides and metal containing anions represent an example of a gas-phase ion/ion reaction that is sensitive to polypeptide structure. These observations are noteworthy in that they allude to the possibility of obtaining information, without requiring fragmentation of the peptide backbone, about ion structure as well as the relative ion affinities associated with the reactants.     

Fragmentation and charge transfer in gas-phase complexes of divalent metal ions with acetonitrile

The development of electrospray has enabled generation of gas-phase multiply charged metal ion complexes with various solvent molecules. These species exhibit rich fragmentation chemistry, involving competition among neutral ligand loss, ligand cleavage, and dissociative electron and proton transfer. Acetonitrile is a common aprotic solvent. Here we present a comprehensive MS/MS study on acetonitrile complexes of divalent metal cations. We measured the critical sizes below which dissociation channels other than the trivial neutral evaporation become operative and minimum sizes at which dications remain stable against charge reduction. For all sizes between the two, low-energy fragmentation patterns have been elucidated in detail.     

Substituent effects on the gas-phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.     

Declustering and fragmentation of protein ions from an electrospray ion source

A triple quadrupole mass spectrometer with a high pressure collision cell has been used to explore the declustering and fragmentation processes that may occur in the vacuum interface region of an electrospray or ionspray ion source. Using apomyoglobin as a model protein compound, collisional processes in Q2 were used to elucidate possible mechanisms which could occur in the orifice-skimmer region to affect the observed charge state distribution. The results indicate that charge loss or gain through collisional loss of a proton or electron does not occur; rather, higher collision energy results in better declustering of lower charge state ions, and fragmentation of higher charge state ions. The net result is an apparent shift toward lower charge state as the collision energy in the free jet region is increased. In addition, the data suggest that a mixture of heavily clustered monomers and possibly dimers and multimers are present in the expansion from ion source into vacuum, and it is this mixture which is acted on by the declustering field to produce the observed mass spectrum. The presence of these “superclusters” needs to be considered in any theory of ion desorption and transport processes in the source and interface region.     

Electrospray ionization mass spectrometry and tandem mass spectrometry of clodronate and related bisphosphonate and phosphonate compounds

The bisphosphonate family with a P-C-P structure is a broad class of drugs, widely investigated as potential inhibitors in bone diseases and calcium metabolic disorders. In this study, the mass spectrometric (MS) behavior and fragmentation of clodronate and related bisphosphonate and phosphonate compounds was studied by using negative ion electrospray ionization (ESI) with triple quadrupole and ion trap instruments. The effect of pH on the degree of deprotonation of the polyprotic bisphosphonic and phosphonic acids in negative ion ESI-MS was investigated, and the degree of deprotonation in the ESI mass spectra and the dissociation in the liquid phase were compared. The results provide evidence that the measured ESI mass spectra do not correlate with the chemistry in the liquid phase owing to the decrease in the pH of the solvent droplets during the ion evaporation process and the charge state neutralization in the gas phase. Ion trap MS(n) provided useful information on the fragmentation study of clodronate and related bisphosphonate and phosphonate compounds, in which interesting fragmentation pathways including the direct elimination of carbon monoxide from deprotonated bisphosphonates and formation of a P-P bond were observed. Reactions between the product ions with a -PO(2) group and residual water in the ion trap or in the high-pressure region of the triple quadrupole instrument formed other unexpected fragmentation paths for all the bisphosphonates studied.     

Multiply charged ions of ruthenium(II), rhodium(III) and cobalt(III) complexes in electrospray ionization mass spectrometry

Multiply charged ions from electrospray ionization (ESI) were observed for ruthenium-bidentate ligand complexes, such as [RuL₂B]X₂ and [(RuL₂)₂B]X₄, where L is 2,2′-bipyridine, B are tetradentate ligands of 2,2′-bis(2′-pyridyl)bibenzimidazole and 2,6-bis(2′-pyridyl)benzodiimidazole, bidentate ligand of 2-(2′-pyridyl)benzimidazole and related compounds and X is CIO₄^- or CI^-. ESI mass spectra showed a simple mass pattern for easy structural assignment and detecting impurities. The mass spectra for binuclear complexes provide a charge state distribution ranging from 4+ to 2+ for Ru(II)—Ru(II) compounds and 5+ to 2+ for Ru(II)—Rh(III) compounds. It was found that different multiply charged ions are generated by loss of counterions and by protonation/deprotonation at the proton site of ligands B. The abundances of these ions are qualitatively explained in terms of the acidity of metal complexes depending on the bridging ligand structures and the charge of the metal ions. Ions produced by removal of ligands were hardly observed.     

Primary to quaternary protein structure determination with electrospray ionization and magnetic sector mass spectrometry

Electrospray ionization with a forward-geometry magnetic sector mass spectrometer was used for collisionally activated dissociation studies of multiply charged polypeptides and for studying non-covalently bound protein systems. The high-resolution capabilities of a high-performance instrument allow the resolution of isotopic contributions for product ions and molecular ion species. Determination of product ion charge states by this method reduces difficulties in the interpretation of product ion mass spectra from multiply charged precursors, which are generated either in the atmospheric pressure/vacuum electrospray interface or in the collision chamber of the mass spectrometer. Extended tandem mass spectrometric experiments have the potential for sequencing larger polypeptides. However, evidence for isomerization of gas-phase product ions from substance P and substance P analogues was observed, complicating the interpretation of product ion spectra. Non-covalent complexes can also be studied by electrospray ionization magnetic sector MS. The higher m/z range of such an instrument is a major advantage for studying weakly bound systems, such as heme–protein systems (myoglobin, hemoglobin) and protein aggregates (concanavalin A), because of their tendency to form complex ions with relatively low charge states.     

Tandem mass spectrometry of half-generation PAMAM dendrimer anions

Ions derived from negative electrospray ionization of polyamidoamine (PAMAM) dendrimer generation 0.5 were subjected to ion trap tandem mass spectrometry. Ion/ion proton transfer reactions were used to manipulate the charge states of PAMAM precursor ions to form lower charge states from those initially formed by electrospray, as well as to facilitate the interpretation of the product ion mass spectra. Most of the products derived from dendrimer precursor ions could be rationalized by retro-Michael decomposition reactions. The dominant fragmentation channels are highly dependent on the composition of the counter-ions, which in this case are restricted to different numbers of sodium ions and protons, and whether the precursor ion is multiply charged or singly charged. An interpretation is given that is consistent with all of the observations made with the various anions associated with this study. The nature of the structural information that can be obtained via ion trap tandem mass spectrometry of the dendrimers is dependent on the types of precursor ions subjected to study. The tandem mass spectrometry data also provided information about the structure of faulty synthesis products present in the PAMAM dendrimer sample.     

Electrospray-assisted laser desorption/ionization and tandem mass spectrometry of peptides and proteins

We have constructed an electrospray-assisted laser desorption/ionization (ELDI) source which utilizes a nitrogen laser pulse to desorb intact molecules from matrix-containing sample solution droplets, followed by electrospray ionization (ESI) post-ionization. The ELDI source is coupled to a quadrupole ion trap mass spectrometer and allows sampling under ambient conditions. Preliminary data showed that ELDI produces ESI-like multiply charged peptides and proteins up to 29 kDa carbonic anhydrase and 66 kDa bovine albumin from single-protein solutions, as well as from complex digest mixtures. The generated multiply charged polypeptides enable efficient tandem mass spectrometric (MS/MS)-based peptide sequencing. ELDI-MS/MS of protein digests and small intact proteins was performed both by collisionally activated dissociation (CAD) and by nozzle-skimmer dissociation (NSD). ELDI-MS/MS may be a useful tool for protein sequencing analysis and top-down proteomics study, and may complement matrix-assisted laser desorption/ionization (MALDI)-based measurements.     

Anisole, a new dopant for atmospheric pressure photoionization mass spectrometry of low proton affinity, low ionization energy compounds

Atmospheric pressure photoionization (APPI) is a novel method of ionization in liquid chromatography/mass spectrometry (LC/MS). It was originally developed in order to broaden the range of LC/MS ionizable compounds towards less polar compounds that cannot be analyzed by electrospray (ESI) and atmospheric pressure chemical ionization (APCI). Studies done thus far have shown that non-polar compounds that earlier were not ionizable in LC/MS can indeed be ionized by the use of APPI. However, the best ionization efficiency for low polarity samples has been achieved with low proton affinity (PA) solvents that are not suitable in reversed-phase LC (RP-LC). Here it is demonstrated that the signals for analytes with low proton affinities in acetonitrile can be increased 100-fold by using anisole as the dopant for APPI, which takes the sensitivity to the same level achieved in the analysis of high PA analytes.     

Apparent gas-phase acidities of multiply protonated peptide ions: Ubiquitin,insulin B,and renin substrate

The gas-phase deprotonation reactions of multiply protonated bovine ubiquitin, insulin chain B, and renin substrate tetradecapeptide ions have been studied in a Fourier transform ion cyclotron resonance mass spectrometer coupled with an external electrospray source. Rate constants were measured for the reactions of these peptide ions with a series of reference compounds of known gas-phase basicities ranging from 195.6 to 232.6 kcal/mol. The apparent gas-phase acidities (GA_app) of the multiply protonated peptide ions [M + nH]ⁿ⁺ were determined with deprotonation reactions. The deduced values of GA_app show a strong dependence on the charge states of the multiply protonated peptide ions. In general, the values decrease as the charge states of the peptide ions increase. For ubiquitin ions, the determined GA_apps values decrease from> 232.6 to 205.0 kcal/mol for n = 4-13; for insulin B ions, the GA_apps decrease from> 232.6 to 198.2 kcal/mol for n = 2-5; for renin substrate ions, the GA_apps decrease from 221.6 to < 195.6 kcal/mol for n = 2-4. Interestingly, at a given mass-to-charge ratio, the GA_apps of these peptide ions agree within 10 kcal/mol despite large differences in their mass and charge. The ubiquitin and insulin B ions generated under the present conditions reveal multiple isomers at certain charge states,n = 4, 5, 6, 12 for ubiquitin and n = 4, 5 for insulin B, as evidenced by the fact that the isomers display distinctively different deprotonation reaction rates with certain reference compounds.     

An analytical strategy for identification of a somatotropin‐like bioactive peptide by ion trap liquid chromatography/electrospray ionization tandem mass spectrometry after immuno‐affinity purification from buffalo serum

The use of growth hormones, such as native and recombinant somatotropins, is forbidden in the European Union (EU), but is legal in the USA. The misuse of recombinant bovine somatotropin in Italy is suspected for enhancing milk production, thanks to its availability on the illegal market. A synthetic bioactive peptide of 27 amino acids derived from bovine somatotropin was successfully tested in France and in southern Italy for scientific purposes, to stimulate milk production, both in cows and buffaloes. This somatotropin‐like peptide (PEP‐ST), suspected for illegal use in southern Italy, was synthesized by linking the 104–113 sequence of bovine somatotropin to the 323–339 sequence of ovalbumin. Herein, a method for detection and identification of the PEP‐ST in buffalo serum is described; our strategy was based on the production of IgG anti‐PEP‐ST, used to synthesize an immuno‐affinity column for peptide purification from buffalo serum, prior to analysis by ion trap liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS). The immuno‐affinity column was successfully used to purify in a single step the bioactive PEP‐ST from buffalo serum samples spiked at 20, 50 and 200 µg/mL for confirmatory analysis. Ion trap LC/ESI‐MS/MS identification was based on detection of a multi‐charged molecular ion and its characteristic fragmentation pattern. No significant matrix interference was observed, accounting for method specificity. We consider this strategy to be a basic approach that could be improved in the perspective of the official control of illegal use of somatotropin and somatotropin‐like compounds in buffalo breeding. Copyright © 2009 John Wiley & Sons, Ltd.     

Gas-phase proton transfer reactions involving multiply charged cytochrome c ions and water under thermal conditions

Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H]¹⁵⁺ ion being the most intense peak. With the addition of H₂O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.     

Mechanism of charging and supercharging molecules in electrospray ionization

The origin of the extent of charging and the mechanism by which multiply charged ions are formed in electrospray ionization have been hotly debated for over a decade. Many factors can affect the number of charges on an analyte ion. Here, we investigate the extent of charging of poly(propyleneimine) dendrimers (generations 3.0 and 5.0), cytochrome c, poly(ethylene glycol)s, and 1,n-diaminoalkanes formed from solutions of different composition. We demonstrate that in the absence of other factors, the surface tension of the electrospray droplet late in the desolvation process is a significant factor in determining the overall analyte charge. For poly(ethylene glycol)s, 1,n-diaminoalkanes, and poly(propyleneimine) dendrimers electrosprayed from single-component solutions, there is a clear relationship between the analyte charge and the solvent surface tension. Addition of m-nitrobenzyl alcohol (m-NBA) into electrospray solutions increases the charging when the original solution has a lower surface tension than m-NBA, but the degree of charging decreases when this compound is added to water, which has a higher surface tension. Similarly, the charging of cytochrome c ions formed from acidified denaturing solutions generally increases with increasing surface tension of the least volatile solvent. For the dendrimers investigated, there is a strong correlation between the average charge state of the dendrimer and the Rayleigh limiting charge calculated for a droplet of the same size as the analyte molecule and with the surface tension of the electrospray solvent. A bimodal charge distribution is observed for larger dendrimers formed from water/m-NBA solutions, suggesting the presence of more than one conformation in solution. A similar correlation is found between the extent of charging for 1,n-diaminoalkanes and the calculated Rayleigh limiting charge. These results provide strong evidence that multiply charged organic ions are formed by the charged residue mechanism. A significantly smaller extent of charging for both dendrimers and 1,n-diaminoalkanes would be expected if the ion evaporation mechanism played a significant role.     

Reactions of poly(ethylene glycol) cations with iodide and perfluorocarbon anions

Multiply charged poly(ethylene glycol) ions of the form (M+nNa) ⁿ⁺ derived from electrospray ionization have been subjected to reactions with negative ions in the quadrupole ion trap. Mixtures of multiply charged positive ions ranging in average mass from about 2000 to about 14,000 Da were observed to react with perfluorocarbon anions by either proton transfer or fluoride transfer. Iodide anions reacted with the same positive ions by attachment. In no case was fragmentation of the polymer ion observed. In all cases, the multiply charged positive ion charge states could be readily reduced to +1, thereby eliminating the charge state overlap observed in the normal electrospray mass spectrum. With all three reaction mechanisms, however, the +1 product ions were comprised of mixtures of products with varying numbers of sodium ions, and in the case of iodide attachment and fluoride transfer, varying numbers of halogen anions. These reactions shift the mass distributions to higher masses and broaden the distributions. The extents to which these effects occur are functions of the magnitudes of the initial charges and the width of the initial charge state distributions. Care must be taken in deriving information about the polymer molecular weight distribution from the singly charged product ions arising from these ion/ion reactions. The cluster ions containing iodide were shown to be intermediates in sodium ion transfer. Dissociation of the adduct ions can therefore lead to a +1 product ion population that is comprised predominantly of M+Na⁺ ions. However, a strategy based on the dissociation of the iodide cluster ions is limited by difficulties in dissociating high mass-to-charge ions in the quadrupole ion trap.     

Use of flow injection atmospheric pressure photoionization quadrupole time-of-flight mass spectrometry for fast olive oil fingerprinting

The recently introduced technique of an atmospheric pressure photoionization (APPI) source coupled to quadrupole time-of-flight mass spectrometry (QqTOFMS) has been applied to fast olive oil fingerprinting on the basis of the accurate mass measurements obtained with this instrumentation. The key compounds can be characterized as [M+H]+ (produced by proton transfer) or as [M]+* (by charge transfer) ions in the mass spectra. [M+H]+ ions, however, show higher abundance, especially for triacylglycerols. Other ions present in APPI-MS are the acylium ion [RiCO]+ and [RiCO-H2O]+. This latter ion is absent in the electrospray ionization (ESI)-MS spectra, and this represents valuable complementary information. Several critical parameters in the APPI source were optimized such as LC eluent composition, ion spray voltage and, especially, declustering potential. APPI-QqTOFMS allows easy discrimination among different edible oils: olive, extra virgin olive, olive-pomace, hazelnut, sunflower, corn and several mixed oils, with high throughput (approximately 1 min per sample). Cluster analysis was applied to obtain the best experimental conditions for oil discrimination on the basis of declustering potential. Principal components analyses of these APPI-MS spectra show that the approach can be used for studies of olive oil adulteration with other oils, even in the case of hazelnut oil that exhibits a high chemical similarity with olive oil.