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Electrospray Ionization Mass Spectrometry☆
Electrospray Ionization Mass Spectrometry☆ WMA Niessen, hyphen MassSpec, Warmond, The Netherlands ã 2016 Elsevier Inc. All rights reserved.
Introduction Principle of Operation History of Electrospray Ionization Instrumentation Practical Aspects Ion-Source Parameters Solvent Composition Electrolyte Additives Detergents Sample Interferences Concluding Remarks Methodologies Based on Electrospray Ionization Applications References
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Introduction In the past twenty years, electrospray ionization (ESI) has become one of the most important ionization methods in mass spectrometry (MS). It is the method-of-choice in the coupling of liquid chromatography and mass spectrometry (LC–MS). It is currently used in over 95% of all LC–MS applications. It is especially useful in the analysis of highly polar, ionic, and macromolecular analytes. In addition, electrospray ionization plays an important role in the characterization of biomacromolecules, especially peptides and proteins. History, principle, instrumentation, practical aspects, and applications of electrospray ionization are discussed in this chapter.
Principle of Operation The use of electrospray nebulization and ionization in MS is based on atmospheric-pressure ionization. The column effluent from reversed-phase LC, ie, a solvent mixture of methanol or acetonitrile and up to 10 mmol/L aqueous buffer, is nebulized into an atmospheric-pressure ion source region. Because pure electrospray nebulization can only be achieved at flow-rates up to 10 mL/min, pneumatically-assisted electrospray is performed in most cases: the liquid flow of 0.1–1.0 mL/min is nebulized into small droplets by a combined action of a strong electric potential between needle and counter electrode, eg, 2–5 kV, and a highspeed concurrent nitrogen flow (50–100 L/h). The electrospray nebulization process results in the formation of small droplets, positively-charged when the source is operated in positive-ion mode and negatively-charged in the negative-ion mode. In their flight between needle and counter electrode, neutral solvent molecules will evaporate from the surface of the charged droplets. The solvent evaporation results in a decreasing droplet size, which in turn leads to shorter distances between the charges at the surface of the droplets. When the surface tension of the liquid can no longer accommodate the Coulomb repulsion between the charges, the droplets disintegrates in a so-called Coulomb explosion or field-induced electrohydrodynamic disintegration process. This leads to the emission of a number of microdroplets with a radius of less than 10% of that of the initial droplets. Gas-phase analyte ions are generated from these microdroplets by a variety of processes, including ion evaporation, soft desolvation, and chemical ionization at the droplet surface (see below). Subsequently, the resulting gas-phase ions can be mass analyzed.
History of Electrospray Ionization Although electrospray nebulization was already described in 1917 by Zeleny, the first applications of electrospray in mass spectrometry date from the late-1960s, when the group of Dole investigated the transfer of macromolecules from the liquid phase to the gas phase by electrospraying dilute solutions in a nitrogen bath gas.1 Their hypothesis was that gas-phase ions from ☆
Change History: January 2016. WMA Niessen updated the complete text of this article, which was originally written in 2005. The discussion on matrix effects and their characterization has been updated. A section on other ways to perform electrospray ionization, ie, via desorption electrospray ionization and paperspray ionization, has been added. A more extensive series of references for further reading have been added and implemented in the text.
Reference Module in Chemistry, Molecular Sciences and Chemical Engineering
http://dx.doi.org/10.1016/B978-0-12-409547-2.12211-5
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macromolecules can be produced by desolvation of the charged droplets produced in electrospray, assuming these macromolecules exist as preformed ions in solution, eg, positively-charged proteins at a pH below their isoelectric point. This ionization mechanism is nowadays called the charge residue model. Next to the work of Dole, where the sample solutions were sprayed in an atmospheric-pressure region, a variety of other related approaches have been described. In the mid-1970s, the group of Evans investigated the applicability of electrospraying solutions in a vacuum.2 The method is called electrohydrodynamic ionization. As the result of the interaction of a strong electrostatic field with a liquid meniscus at the end of a capillary tube, solvated ions are emitted from the apex of a so-called Taylor cone, formed at the capillary tip. Due to the high-vacuum conditions, the use of non-volatile organic solvents, eg, glycerol doped with sodium iodide to increase the conductivity, is required for analyte ionization. Compounds investigated are saccharides, nucleosides, and small peptides. Ions are formed by attachment of a cation or an anion to the analyte molecules. In 1980, the group of Zolotai reported similar experiments using glycerol or water as solvent and introduced the term ’field evaporation of ions from solution’.3 An important contribution to this field was made by Iribarne and Thomson in the late-1970s, who investigated the processes of the direct emission of ions from liquid droplets.4 In their experimental setup, a liquid solution is pneumatically nebulized in an atmospheric-pressure chamber and the droplets produced are charged by random statistical charging using an induction electrode positioned close to the nebulizer. Solvated single-charge ions are formed in the evaporating spray, which are sampled into a differentially pumped quadrupole mass spectrometer. Their theoretical description of the ionization process, ie, the so-called ion evaporation model, is adapted later by researchers in the field of thermospray and electrospray ionization. The ion formation in thermospray ionization, introduced in the early-1980s by Vestal and coworkers,5 was explained in terms of ion evaporation. Preformed analyte ions are evaporated from small, fast-evaporating, charged droplets generated by the thermospray nebulization process. Although the importance of ion evaporation in thermospray ionization is questioned, the emphasis put at it at the time certainly stimulated further investigation into liquid-based ionization approaches for mass spectrometry. Within this context, Yamashita and Fenn6,7 in the mid-1980s continued the original ESI experiments of Dole and implemented molecular beam technology to solve some of the experimental difficulties experienced by Dole. The liquid is electrosprayed into a bath gas. The dispersion of ions, solvent vapor and bath gas is expanded into a vacuum chamber, forming a supersonic jet, the core of which is sampled into the mass spectrometer by means of a skimmer. Initially, they tried in vain to reproduce the experiments of Dole with biomacromolecules, but nevertheless continued to investigate the sequence of electrospraying and droplet evaporation with low molecular-mass compounds. They concluded that the hypothesis of Dole1 was not valid in their experiment and they recognized the importance of the work of Iribarne and Thomson4 in this field. The major breakthrough in this research was achieved in 1988, when the formation of multiple-charge protein ions was demonstrated (Fig. 1).8,9 This revolutionized the
Fig. 1 First spsectra demonstrating multiply-charged proteins generated by electrospray ionization mass spectrometry. Reproduced from Mann, M.; Meng, C.K.; Fenn, J.B. Anal. Chem. 1989, 61, 1702 with permission of the American Chemical Society.
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application of MS, because biomacromolecules could now be analyzed using simple and relatively low-cost quadrupole mass analyzers. In 2002, Fenn received the Nobel prize for chemistry for his contribution to mass spectrometric analysis and characterization of biomacromolecules. Although Fenn initially very much emphasized the importance of ion evaporation in the ion formation by ESI, extensive research has demonstrated that, depending on the analyte and the experimental conditions, the charge-residue model and the ion-evaporation model are both important.10,11 In addition, especially in the analysis of small molecules, gas-phase ion-molecule reactions appear to play an important role in ion formation by electrospray ionization.
Instrumentation After the first demonstration of multiple-charge gas-phase proteins ions, all major instrument manufacturers developed atmospheric-pressure ion sources, equipped with ESI interfaces for both protein characterization and LC–MS applications. Within five years, ESI interfacing became the method of choice in LC–MS coupling. It lead to a huge increase in the use of MS for the characterization and identification of labile and polar analytes as well as for routine quantitative analysis. The advent of ESI for peptide and protein analysis stimulated further development and analytical application of existing and new mass analysis approaches, such as quadrupole ion traps, Fourier-transform ion-cyclotron resonance MS, quadrupole–time-of-flight hybrid, and orbitrap (hybrid) instruments. It opened new application areas, such as proteomics. LC–MS with ESI and in selected-reaction monitoring (SRM) mode in tandem-quadrupole MS–MS instruments has become the method-of-choice in routine quantitative bioanalysis supporting preclinical and clinical studies within the pharmaceutical industry as well as in many other areas. Since the first introduction of commercial electrospray interfaces for LC–MS, based on the Fenn design,6 continuous developments have taken place, resulting in more reliable and robust as well as more efficient interfaces. Some aspects of these developments are briefly discussed here. An atmospheric-pressure ion source for ESI consists of five parts (Fig. 2): (1) the pneumatically-assisted electrospray needle, used for the introduction of sample solution or LC mobile phase, (2) the actual (atmospheric-pressure) ion source region, where ions are generated from the microdroplets, (3) the ion-sampling orifice, (4) the atmospheric-pressure to high-vacuum interface, and (5) the ion-optical system, where the ions generated in the source are analyte-enriched and transported towards the highvacuum mass analyzer.12 In principle, two types of electrospray interfaces must be distinguished. Most attention is paid to the high-flow devices applied for routine LC–MS. However, an extreme low-flow electrospray needle device, ie, the so-called nanoelectrospray, has been developed as well, especially by Wilm and Mann13 in the mid-1990s. Nanoelectrospray is especially important in the low nL/min introduction of protein and peptide solutions, and is increasingly important in the coupling to MS of micro- and nanoLC and capillary electromigration techniques, such as capillary electrophoresis and isoelectric focusing. Many precautions described below for the high-flow devices are not necessary in the low-flow devices. Different instrument manufacturers have developed a wide variety of ion source and interface designs. Initially, the electrospray needle was positioned axial with the ion-sampling orifice. Due to the heavy use of electrospray LC–MS in high-throughput quantitative bioanalysis, alternative positions had to be investigated in order to prevent a rapid contamination of the ion-sampling orifice by non-volatile contaminants in the liquid stream. The liquid-introduction needle is nowadays positioned orthogonal to the ion-sampling orifice. In addition, a countercurrent nitrogen flow is applied around the ion-sampling orifice to further protect it against precipitation of non-volatiles. The increase in the flow-rate of the liquid introduced by means of pneumatically-assisted electrospray required the supply of heat to the spray in order to stimulate the solvent evaporation from
Fig. 2 Schematic diagram of an atmospheric-pressure ionization interface.
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the droplets. In most systems, this is achieved by the application of heated nitrogen streams, either concurrent, orthogonal to the spray, like in turbo-ionspray, or countercurrent. In addition, the ion source block and ion-sampling orifice is heated. The glass capillary with two-sided conductive layers, as introduced by Fenn,6 is still applied as ion-sampling device in some of the commercial interfaces. However, a variety of other ion-sampling devices have been introduced, including orifices in flat plates and sample cones as well as heated stainless-steel capillaries. While initially, only one differentially pumped stage between atmospheric-pressure ion source and mass analyzer was applied,6 two or even three pumping stages are used in current systems. Significant effort has been put in increasing the pumping speed at the transition zone, partly in order to increase the size of the ion-sampling orifice. Significant progress has been made in turbomolecular pumps, enabling the use of small and relatively efficient dual-inlet pumps in small benchtop LC–MS systems. Skimmers are used between the various pumping regions. In the relatively large pumping regions, evacuated by turbomolecular pumps, a variety of RF-only multipole devices are used, eg, quadrupoles, hexapoles, octapoles, and ion tunnels. These devices are used to transport the ions through these pumping regions with the highest possible ion transmission and to focus the ions into the mass analyzer.
Practical Aspects Successful operation of the electrospray interface requires the optimization of a variety of interrelated ion-source parameters. In addition, the solvent composition and flow-rate may have influence on the system performance.
Ion-Source Parameters The ion-source parameters and their optimum values depend on the specific ESI source applied, eg, in some source designs the optimum voltage difference between needle and counter electrode is around 4–5 kV, while in other designs the optimum is around 2–3 kV. Most systems nowadays allow for an automatic software-controlled optimization of the voltages in the source and in the mass spectrometer. These procedures search for optimum ion production and transmission towards the detector. In addition, the settings for SRM transitions, to be used in quantitative analysis can be optimized in this way. An SRM transition is a combination of precursor ion and product ion and the optimum instrument parameters to measure these ions. Although very useful, the procedures do not account for the important influence of mobile-phase composition and sample or matrix effects on the performance of the electrospray interface.
Solvent Composition Both the solvent composition and interfering contaminants in the sample solution can have distinct influence on the efficiency of ESI for a particular analyte. In general, reversed-phase type mobile phases are applied, ie, mixtures of water and either methanol or acetonitrile containing an acid, base, or buffer. It is often found that most analytes show better response from solutions containing > 50% organic solvent (methanol or acetonitrile in most cases). However, ESI is generally not possible from pure organic solvents. Obviously, the organic content of the mobile phase in LC–MS is more determined by retention characteristics in LC than by ionization and MS detection. It has been demonstrated for a large variety of compounds that a methanol-containing mobile phase leads to a better response than an acetonitrile-containing mobile phase. Post-column and/or sheath-flow addition of high-organic solvents, such as aqueous mixtures with 2-propanol or methoxyethanol, are sometimes applied, especially in the analysis of proteins by ESI-MS.
Electrolyte Additives The composition, pH, and concentration of the buffer used also influences the performance of the ESI interface. Based on the ionization mechanisms, briefly outlined above, one would conclude that an acidic mobile phase is needed for the analysis of an organic base and a basic solution for an organic acid. In this way, the formation of liquid-phase preformed ions is promoted. However, in the ESI-MS analysis of especially small molecules (<1 kDa), the picture appears to be more complex. Wrong-wayaround electrospray has been described, where organic bases are ionized from basic solutions in positive-ion mode without significant loss in response.14 Although most of the current LC–MS interfaces can readily cope with non-volatile mobile-phase additives such as sodium phosphate buffers, they should not be used in routine LC–MS applications. They may lead to more pronounced adduct formation, ie, formation of [M + Na]+ next to [M + H]+, and may lead to significant suppression of the response. Therefore, the use of volatile additives is highly recommended. Mallet and coworkers15 investigated the influence of a number of mobile-phase additives on the response of eight basic drugs in positive-ion mode and eight acidic drugs in negative-ion mode. The influence of formic, acetic, and trifluoroacetic acid, and of ammonium hydroxide, formate, biphosphonate and bicarbonate was investigated as a function of additive concentration. Some of their results are pictured in Fig. 3. The ionization of all compounds, both basic and acidic ones, was found to be suppressed by the addition of strong ion-pairing agents like trifluoroacetic acid or nonafluoropentadecanoic acid. Most compounds respond similarly to the addition of acetic acid and formic acid, although the effect is compound dependent. In positive-ion mode, some compounds
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Fig. 3 Effect of additives, formic acid (FA), trifluoroacetic acid (TFA), ammonium hydroxide (NH4OH), and acetic acid (AcOH) on the relative response of (A) propranolol, and (B) risperidone in electrospray ionization (compared to 50% aqueous methanol, which is 100%). Reproduced from Mallet,C.R.; Lu,Z.; Mazzeo, J.R. Rapid Commun. Mass Spectrom., 2004, 18, 49.
(propranolol, trimethoprim, terfenadine, methoxyverapamil, and reserpine) show ionization enhancement at acid concentrations below 0.5%, while others show ionization suppression (risperidone and benextramine). Somewhat surprisingly, the response of some bases (risperidone, terfenadine, metoxyverapamil, benextramine) are enhanced by addition of ammonium hydroxide. The quaternary ammonium compound pipenzolate is not significantly influenced by additions of ammonium hydroxide or formic or acetic acid. Most basic compounds are suppressed by the addition of ammonium formate, bicarbonate and biphosphonate. In negative-ion mode, the ionization of most compounds is suppressed by formic and acetic acid additives. Ammonium hydroxide results in (significant) ionization enhancement for cholic acid, raffinose, canrenoic acid, while the ionization of fumaric acid, malic acid, and etidronic acid is strongly suppressed. The response of cholic acid, raffinose, and canrenoic acid is also enhanced by ammonium formate and bicarbonate. Ammonium biphosphate suppresses the response of all acidic compounds.
Detergents The influence of various detergents on the electrospray ionization of proteins was also investigated in some detail.16 The detergents are frequently required for the solubilization of the proteins. At concentrations of 0.01% or below, only sodium dodecylsulfate and taurocholate show significant ionization suppression. At 0.1%, most of the detergents result in significant suppression, eg, to below 10% for sodium dodecylsulfate and taurocholate, CTAB, LDAO, and Triton X-100, to the 10–30% range for sodium cholate, and various alkyl glucosides, and to the 30–60% range for CHAPS, hexyl and dodecyl glucosides.
Sample Interferences In quantitative analysis, further ionization suppression or enhancement may be due to co-eluting sample constituents not removed by the sample pre-treatment. This is often indicated as a matrix effect. In most cases, the identity of the components causing the matrix effects is not clear. It has been demonstrated that non-volatile residues in the pre-treated plasma or urine samples and
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especially phospholipids in plasma samples are important causes of matrix effects. If the ionization suppression is not too severe, ie, not compromising the method detection limit too much, the absolute matrix effect would not be of significant importance. However, it is found that a relative matrix effects can occur as well, which are non-reproducible ionization suppression or enhancement effects between different batches of the same sample. Thus, method development for quantitative analysis should include studies on multiple sample batches in order to study relative matrix effects.17,18 To some extent, matrix effects can be overcome by the use of stable-isotope-labelled internal standards (SIL-IS), although results have been reported where SIL-IS and analyte experience different matrix influences. When in method development and validation a matrix effect is observed which compromises the reliability of the method (poor %RSD), further attention must be paid to improving the sample pre-treatment method. The best way to study matrix effects involves the analysis of calibration series of three sets samples: (1) solvent standards containing analyte and SIL-IS, (2) postextraction spiked matrix samples containing analyte and SIL-IS, and (3) pre-extraction spiked matrix samples containing analyte and SIL-IS. Comparison of peak areas (or peak area ratios) between post-extracted spiked samples and solvent standards allows the assessment of the absolute matrix effect. From the peak areas (or peak area ratios) of pre-extraction and post-extraction spiked matrix samples, the extraction recovery can be determined. If samples from a number of independent matrix batches, eg, 5–6 batches, are analysed, the absolute differences of the slopes and the relative standard deviation of the slopes of the calibration plots obtained can be used to assess the relative matrix effects.17,18
Concluding Remarks Being a liquid-phase ionization method, the performance of ESI can be significantly influenced by the solvent composition, especially mobile-phase additives and sample interferences. Nevertheless, ESI-MS can be successfully applied in the (quantitative) analysis of samples from various application areas. In most cases, the method development for LC–MS is easier to perform than for LC–UV. However, one should keep in mind that these two detection techniques respond differently to interfering compounds, and therefore require a different method development and optimization strategies. With proper validation, LC–MS is a powerful approach to quantitative analysis. Care must be taken in the selection of mobile-phase additives and in sample pre-treatment.
Methodologies Based on Electrospray Ionization ESI-MS has become an important tool in analytical chemistry. It is widely applied in LC–MS in many application areas (see below). However, the power of ESI-MS in analyte ionization has also led to a range of derived methods where analyte ionization by ESI is achieved in different experimental setups, not necessarily involving continuous solvent delivery like in LC–MS. The nanoelectrospray interface, discussed above,13 can be applied to couple nano-LC to MS, but also to perform the MS analysis of small samples ( 1 mL) from a nanoelectrospray needle. With flow-rates in the range of 10 nL/min, this allows to perform a range of MS and tandem mass spectrometry (MS–MS) experiments. A different approach to sample analysis is provided by desorption electrospray ionization (DESI).19 In DESI, a high-velocity spray of charged microdroplets from a (pneumatically-assisted) electrospray needle is directed at a surface, mounted in front of the ion-sampling orifice of an API source. DESI releases gas-phase ions from the surface material or surface constituents, which can be analyzed by MS. DESI enables the study of compounds at surfaces without extensive isolation and sample pre-treatment steps, eg, drugs of abuse from tablets, and also opens possibilities to perform chemical imaging of surfaces such as thin-layer chromatography plates and tissue sections.20 In paperspray ionization (PSI), biomolecules are ionized from a paper tip emerged with non-polar solvents like hexane or toluene, placed in the electric field close to the ion-sampling orifice of an API source.21
Applications The advent of ESI-MS certainly opened many new application areas for MS and LC–MS analysis. This is based on the ability to provide extreme soft liquid-based ionization. The most important application area is perhaps the analysis of peptides and proteins. The possibility to perform rapid molecular-weight determination of proteins up to 200 kDa and beyond stimulated the commercial availability of MS instrumentation featuring atmospheric-pressure ion sources, equipped with ESI. Other application areas benefited from these developments. LC–MS has become an important analytical tool in many areas of drug development within the pharmaceutical industry, in the study of natural products in plants and animals, in food and environmental analysis and in the clinical application area for therapeutic drug monitoring, profiling of endogenous compounds for diagnostic purposes, systematic toxicological analysis, and monitoring of inherited metabolic diseases. As indicated, ESI-MS plays an important role in the characterization of proteins. In this area, ESI-MS is complemented by matrixassisted laser desorption ionization MS (MALDI-MS), which in some cases appears to be less prone to ionization suppression effects by sample constituents and/or additives. First of all, the formation of multiple-charge ions of proteins enables the accurate determination of the protein molecular weight, generally within 0.1% accuracy. The ion envelope of multiple-charge proteins ions can actually be a disadvantage, especially in the analysis of proteins with significant heterogeneity due to for instance posttranslational glycosylation. In current protein research, the protein, or even a complete proteome, is often enzymatically digested to
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Fig. 4 Negative ion electrospray ionization mass spectrum of calmodulin (50 mM) in the presence of melittin (50 mM) and 100-mM calcium acetate, pH 5.7. Ions in the range between m/z 900 and 1600 represent uncomplexed calmodulin and ions in the range between m/z 1700 and 3000. represent the noncovalent calmodulin/melittin complex. Reproduced from Veenstra, T.D. et al., J. Am. Soc. Mass Spectrom. 1998, 9, 580–584 with permission from Elsevier Science.
a peptide mixture. The MS characterization of this mixture leads to a peptide map. Due to its ability to produced single-charge ions, MALDI-MS is preferred in most cases in this initial research. Data from the peptide map are applied to bioinformatics tools involving a protein database search in an attempt to identify the protein. If further studies are required to identify the protein, eg, peptide sequencing by means of MS–MS, the use of ESI is often more favorable, because it is coupled more readily to tandemquadrupole, ion-trap, or quadrupole–time-of-flight and orbitrap hybrid instruments. The identification and quantification of proteins in complex biological samples is especially important in the current proteomics research. While for many years MALDI-MS was the method of choice, because of the ease with which it is combined to two-dimensional gel electrophoresis, the role of ESI-MS in combination with multidimensional LC is increasing. Already in the early applications of ESI-MS in the analysis of proteins it was found that ESI can significantly contribute to the study of protein conformation in solution. While initially solvent-induced conformational changes were investigated, such studies were extended by the use of hydrogen/deuterium exchange and other gas-phase reactions. More recently, ion-mobility spectrometry was implemented in MS–MS instruments as a powerful tool in the assessment of conformational changes of proteins. Such tools are not only applied in fundamental studies, but also applied to detection and monitoring of changes in protein conformation related to for instance neurodegenerative diseases like Alzheimer, Parkinson and Creutzfeldt-Jacobs disease. Another important application area of ESI-MS is the study of noncovalent complexes between proteins and inhibitors, cofactors, metal ions, carbohydrates, other peptides and proteins, substrates, and of nucleic acid complexes. In most cases such complexes exhibit relatively low charge states, resulting in high m/z. In most cases, these so-called native-MS studies22 of such complexes requires the use of either modified quadrupole mass analyzers, to enlarge their m/z range, or time-of-flight instrument, which show an inherent larger m/z range. For example, the group of Naylor23 examined the binding of the calcium-binding protein calmodulin to the calmodulin-dependent protein kinase II (CamK-II) and to melittin. Both peptides form equimolar complexes with calmodulin only in the presence of calcium (stoichiometry was 1: 1: 4 for calmodulin: peptide: Ca). A typical spectrum of the calmodulin/melittin complex is shown in Fig. 4. These type of studies are relevant both in the field of structural biology and in the field of drug discovery, enabling the study of drug-protein or inhibitor-protein interactions.
References 1. Dole, M.; Hines, R. L.; Mack, L. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. Molecular Beams of Macroions. J. Chem. Phys. 1968, 49, 2240–2249. 2. Stimpson, B. P.; Evans, C. A., Jr. Electrohydrodynamic Ionization Mass Spectrometry of Biochemical Materials. Biomed. Mass Spectrom. 1978, 5, 52–63. 3. Zolotai, N. B.; Karpov, G. V.; Tal’roze, V. L.; Skurat, V. E.; Basyuta, Yu. V.; Ramendik, G. I. Mass Spectrometry of the Field Evaporation of Ions from Water and Aqueous Solutions, Aqueous Sodium Iodide and Saccharose Solutions. J. Anal. Chem. USSR 1980, 35, 1161–1174. 4. Iribarne, J. V.; Thomson, B. A. On the Evaporation of Small Ions from Charged Droplets. J. Chem. Phys. 1976, 64, 2287–2294.
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Blakley, C. R.; Vestal, M. L. Thermospray Interface for Liquid Chromatography/Mass Spectrometry. Anal. Chem. 1983, 55, 750–754. Yamashita, M.; Fenn, J. B. Electrospray ion Source. Another Variation of the Free-jet Theme. J. Phys. Chem. 1984, 88, 4451–4459. Fenn, J. B.; Mann, M.; Men, C. K.; Wong, S. F.; Whitehouse, C. M. Electrospray Ionization — Principles and Practice. Mass Spectrom. Rev. 1990, 9, 37–70. Mann, M.; Meng, C. K.; Fenn, J. B. Interpreting Mass Spectra of Multiply Charged Ions. Anal. Chem. 1989, 61, 1702–1708. Covey, T. R.; Bonner, R. F.; Shushan, B. I.; Henion, J. D. The Determination of Protein, Oligonucleotide and Peptide Molecular Weights by ion-Spray Mass Spectrometry. Rapid Commun. Mass Spectrom. 1988, 2, 249–256. Smith, R. D.; Light-Wahl, K. J. The Observation of non-Covalent Interactions in Solution by Electrospray Ionization Mass Spectrometry: Promise, Pitfalls and Prognosis. Biol. Mass Spectrom. 1993, 22, 493–501. Kebarle, P.; Peschke, M. On the Mechanisms by which the Charged Droplets Produced by Electrospray Lead to gas Phase Ions. Anal. Chim. Acta 2000, 406, 11–35. Niessen, W. M. A. Liquid chromatography-mass spectrometry, 3rd ed.; ISBN 978-0-824-74082-5. Wilm, M. S.; Mann, M. Analytical Properties of the Nanoelectrospray ion Source. Anal. Chem. 1996, 68, 1–8. Zhou, A.; Cook, K. D. Protonation in Electrospray Mass Spectrometry: Wrong-way-Round or Right-way-Round? J. Am. Soc. Mass Spectrom. 2000, 11, 961–966. Mallet, C. R.; Lu, Z.; Mazzeo, J. R. A Study of ion Suppression Effects in Electrospray Ionization from Mobile Phase Additives and Solid-Phase Extracts. Rapid Commun. Mass Spectrom. 2004, 18, 49–58. Ogorzalek Loo, R. R.; Dales, N.; Andrews, P. C. Surfactant Effects on Protein Structure Examined by Electrospray Ionization Mass Spectrometry. Protein Sci. 1994, 3, 1975–1983. Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Strategies for the Assessment of Matrix Effects in Quantitative Bioanalytical Methods Based on HPLC–MS–MS. Anal. Chem. 2003, 75, 3019–3030. Matuszewski, B. K. Standard Line Slopes as a Measure of a Relative Matrix Effect in Quantitative HPLC–MS Bioanalysis. J. Chromatogr. B 2006, 830, 293–300. Taka´ts, Z.; Wiseman, J. M.; Cooks, R. G. Ambient Mass Spectrometry using Desorption Electrospray Ionization (DESI): Instrumentation, Mechanisms and Applications in Forensics, Chemistry, and Biology. J. Mass Spectrom. 2005, 40, 1261–1275. Wu, C.; Dill, A. L.; Eberlin, L. S.; Cooks, R. G.; Ifa, D. R. Mass Spectrometry Imaging Under Ambient Conditions. Mass Spectrom. Rev. 2013, 32, 218–243. Li, A.; Wang, H.; Ouyang, Z.; Cooks, R. G. Paper Spray Ionization of Polar Analytes using Non-Polar Solvents. Chem. Commun. 2011, 47, 2811–2813. Lorenzen, K.; van Duijn, E. Native Mass Spectrometry as a Tool in Structural Biology. Curr. Protoc. Protein Sci. 2010, 62, 17.12.1–17.12.17. Veenstra, T. D.; Tomlinson, A. J.; Benson, L.; Kumar, R.; Naylor, S. Low Temperature Aqueous Electrospray Ionization Mass Spectrometry of Noncovalent Complexes. J. Am. Soc. Mass Spectrom. 1998, 9, 580–584.
Introduction Principle of Operation History of Electrospray Ionization Instrumentation Practical Aspects Ion-Source Parameters Solvent Composition Electrolyte Additives Detergents Sample Interferences Concluding Remarks Methodologies Based on Electrospray Ionization Applications References
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Introduction In the past twenty years, electrospray ionization (ESI) has become one of the most important ionization methods in mass spectrometry (MS). It is the method-of-choice in the coupling of liquid chromatography and mass spectrometry (LC–MS). It is currently used in over 95% of all LC–MS applications. It is especially useful in the analysis of highly polar, ionic, and macromolecular analytes. In addition, electrospray ionization plays an important role in the characterization of biomacromolecules, especially peptides and proteins. History, principle, instrumentation, practical aspects, and applications of electrospray ionization are discussed in this chapter.
Principle of Operation The use of electrospray nebulization and ionization in MS is based on atmospheric-pressure ionization. The column effluent from reversed-phase LC, ie, a solvent mixture of methanol or acetonitrile and up to 10 mmol/L aqueous buffer, is nebulized into an atmospheric-pressure ion source region. Because pure electrospray nebulization can only be achieved at flow-rates up to 10 mL/min, pneumatically-assisted electrospray is performed in most cases: the liquid flow of 0.1–1.0 mL/min is nebulized into small droplets by a combined action of a strong electric potential between needle and counter electrode, eg, 2–5 kV, and a highspeed concurrent nitrogen flow (50–100 L/h). The electrospray nebulization process results in the formation of small droplets, positively-charged when the source is operated in positive-ion mode and negatively-charged in the negative-ion mode. In their flight between needle and counter electrode, neutral solvent molecules will evaporate from the surface of the charged droplets. The solvent evaporation results in a decreasing droplet size, which in turn leads to shorter distances between the charges at the surface of the droplets. When the surface tension of the liquid can no longer accommodate the Coulomb repulsion between the charges, the droplets disintegrates in a so-called Coulomb explosion or field-induced electrohydrodynamic disintegration process. This leads to the emission of a number of microdroplets with a radius of less than 10% of that of the initial droplets. Gas-phase analyte ions are generated from these microdroplets by a variety of processes, including ion evaporation, soft desolvation, and chemical ionization at the droplet surface (see below). Subsequently, the resulting gas-phase ions can be mass analyzed.
History of Electrospray Ionization Although electrospray nebulization was already described in 1917 by Zeleny, the first applications of electrospray in mass spectrometry date from the late-1960s, when the group of Dole investigated the transfer of macromolecules from the liquid phase to the gas phase by electrospraying dilute solutions in a nitrogen bath gas.1 Their hypothesis was that gas-phase ions from ☆
Change History: January 2016. WMA Niessen updated the complete text of this article, which was originally written in 2005. The discussion on matrix effects and their characterization has been updated. A section on other ways to perform electrospray ionization, ie, via desorption electrospray ionization and paperspray ionization, has been added. A more extensive series of references for further reading have been added and implemented in the text.
Reference Module in Chemistry, Molecular Sciences and Chemical Engineering
http://dx.doi.org/10.1016/B978-0-12-409547-2.12211-5
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macromolecules can be produced by desolvation of the charged droplets produced in electrospray, assuming these macromolecules exist as preformed ions in solution, eg, positively-charged proteins at a pH below their isoelectric point. This ionization mechanism is nowadays called the charge residue model. Next to the work of Dole, where the sample solutions were sprayed in an atmospheric-pressure region, a variety of other related approaches have been described. In the mid-1970s, the group of Evans investigated the applicability of electrospraying solutions in a vacuum.2 The method is called electrohydrodynamic ionization. As the result of the interaction of a strong electrostatic field with a liquid meniscus at the end of a capillary tube, solvated ions are emitted from the apex of a so-called Taylor cone, formed at the capillary tip. Due to the high-vacuum conditions, the use of non-volatile organic solvents, eg, glycerol doped with sodium iodide to increase the conductivity, is required for analyte ionization. Compounds investigated are saccharides, nucleosides, and small peptides. Ions are formed by attachment of a cation or an anion to the analyte molecules. In 1980, the group of Zolotai reported similar experiments using glycerol or water as solvent and introduced the term ’field evaporation of ions from solution’.3 An important contribution to this field was made by Iribarne and Thomson in the late-1970s, who investigated the processes of the direct emission of ions from liquid droplets.4 In their experimental setup, a liquid solution is pneumatically nebulized in an atmospheric-pressure chamber and the droplets produced are charged by random statistical charging using an induction electrode positioned close to the nebulizer. Solvated single-charge ions are formed in the evaporating spray, which are sampled into a differentially pumped quadrupole mass spectrometer. Their theoretical description of the ionization process, ie, the so-called ion evaporation model, is adapted later by researchers in the field of thermospray and electrospray ionization. The ion formation in thermospray ionization, introduced in the early-1980s by Vestal and coworkers,5 was explained in terms of ion evaporation. Preformed analyte ions are evaporated from small, fast-evaporating, charged droplets generated by the thermospray nebulization process. Although the importance of ion evaporation in thermospray ionization is questioned, the emphasis put at it at the time certainly stimulated further investigation into liquid-based ionization approaches for mass spectrometry. Within this context, Yamashita and Fenn6,7 in the mid-1980s continued the original ESI experiments of Dole and implemented molecular beam technology to solve some of the experimental difficulties experienced by Dole. The liquid is electrosprayed into a bath gas. The dispersion of ions, solvent vapor and bath gas is expanded into a vacuum chamber, forming a supersonic jet, the core of which is sampled into the mass spectrometer by means of a skimmer. Initially, they tried in vain to reproduce the experiments of Dole with biomacromolecules, but nevertheless continued to investigate the sequence of electrospraying and droplet evaporation with low molecular-mass compounds. They concluded that the hypothesis of Dole1 was not valid in their experiment and they recognized the importance of the work of Iribarne and Thomson4 in this field. The major breakthrough in this research was achieved in 1988, when the formation of multiple-charge protein ions was demonstrated (Fig. 1).8,9 This revolutionized the
Fig. 1 First spsectra demonstrating multiply-charged proteins generated by electrospray ionization mass spectrometry. Reproduced from Mann, M.; Meng, C.K.; Fenn, J.B. Anal. Chem. 1989, 61, 1702 with permission of the American Chemical Society.
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application of MS, because biomacromolecules could now be analyzed using simple and relatively low-cost quadrupole mass analyzers. In 2002, Fenn received the Nobel prize for chemistry for his contribution to mass spectrometric analysis and characterization of biomacromolecules. Although Fenn initially very much emphasized the importance of ion evaporation in the ion formation by ESI, extensive research has demonstrated that, depending on the analyte and the experimental conditions, the charge-residue model and the ion-evaporation model are both important.10,11 In addition, especially in the analysis of small molecules, gas-phase ion-molecule reactions appear to play an important role in ion formation by electrospray ionization.
Instrumentation After the first demonstration of multiple-charge gas-phase proteins ions, all major instrument manufacturers developed atmospheric-pressure ion sources, equipped with ESI interfaces for both protein characterization and LC–MS applications. Within five years, ESI interfacing became the method of choice in LC–MS coupling. It lead to a huge increase in the use of MS for the characterization and identification of labile and polar analytes as well as for routine quantitative analysis. The advent of ESI for peptide and protein analysis stimulated further development and analytical application of existing and new mass analysis approaches, such as quadrupole ion traps, Fourier-transform ion-cyclotron resonance MS, quadrupole–time-of-flight hybrid, and orbitrap (hybrid) instruments. It opened new application areas, such as proteomics. LC–MS with ESI and in selected-reaction monitoring (SRM) mode in tandem-quadrupole MS–MS instruments has become the method-of-choice in routine quantitative bioanalysis supporting preclinical and clinical studies within the pharmaceutical industry as well as in many other areas. Since the first introduction of commercial electrospray interfaces for LC–MS, based on the Fenn design,6 continuous developments have taken place, resulting in more reliable and robust as well as more efficient interfaces. Some aspects of these developments are briefly discussed here. An atmospheric-pressure ion source for ESI consists of five parts (Fig. 2): (1) the pneumatically-assisted electrospray needle, used for the introduction of sample solution or LC mobile phase, (2) the actual (atmospheric-pressure) ion source region, where ions are generated from the microdroplets, (3) the ion-sampling orifice, (4) the atmospheric-pressure to high-vacuum interface, and (5) the ion-optical system, where the ions generated in the source are analyte-enriched and transported towards the highvacuum mass analyzer.12 In principle, two types of electrospray interfaces must be distinguished. Most attention is paid to the high-flow devices applied for routine LC–MS. However, an extreme low-flow electrospray needle device, ie, the so-called nanoelectrospray, has been developed as well, especially by Wilm and Mann13 in the mid-1990s. Nanoelectrospray is especially important in the low nL/min introduction of protein and peptide solutions, and is increasingly important in the coupling to MS of micro- and nanoLC and capillary electromigration techniques, such as capillary electrophoresis and isoelectric focusing. Many precautions described below for the high-flow devices are not necessary in the low-flow devices. Different instrument manufacturers have developed a wide variety of ion source and interface designs. Initially, the electrospray needle was positioned axial with the ion-sampling orifice. Due to the heavy use of electrospray LC–MS in high-throughput quantitative bioanalysis, alternative positions had to be investigated in order to prevent a rapid contamination of the ion-sampling orifice by non-volatile contaminants in the liquid stream. The liquid-introduction needle is nowadays positioned orthogonal to the ion-sampling orifice. In addition, a countercurrent nitrogen flow is applied around the ion-sampling orifice to further protect it against precipitation of non-volatiles. The increase in the flow-rate of the liquid introduced by means of pneumatically-assisted electrospray required the supply of heat to the spray in order to stimulate the solvent evaporation from
Fig. 2 Schematic diagram of an atmospheric-pressure ionization interface.
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the droplets. In most systems, this is achieved by the application of heated nitrogen streams, either concurrent, orthogonal to the spray, like in turbo-ionspray, or countercurrent. In addition, the ion source block and ion-sampling orifice is heated. The glass capillary with two-sided conductive layers, as introduced by Fenn,6 is still applied as ion-sampling device in some of the commercial interfaces. However, a variety of other ion-sampling devices have been introduced, including orifices in flat plates and sample cones as well as heated stainless-steel capillaries. While initially, only one differentially pumped stage between atmospheric-pressure ion source and mass analyzer was applied,6 two or even three pumping stages are used in current systems. Significant effort has been put in increasing the pumping speed at the transition zone, partly in order to increase the size of the ion-sampling orifice. Significant progress has been made in turbomolecular pumps, enabling the use of small and relatively efficient dual-inlet pumps in small benchtop LC–MS systems. Skimmers are used between the various pumping regions. In the relatively large pumping regions, evacuated by turbomolecular pumps, a variety of RF-only multipole devices are used, eg, quadrupoles, hexapoles, octapoles, and ion tunnels. These devices are used to transport the ions through these pumping regions with the highest possible ion transmission and to focus the ions into the mass analyzer.
Practical Aspects Successful operation of the electrospray interface requires the optimization of a variety of interrelated ion-source parameters. In addition, the solvent composition and flow-rate may have influence on the system performance.
Ion-Source Parameters The ion-source parameters and their optimum values depend on the specific ESI source applied, eg, in some source designs the optimum voltage difference between needle and counter electrode is around 4–5 kV, while in other designs the optimum is around 2–3 kV. Most systems nowadays allow for an automatic software-controlled optimization of the voltages in the source and in the mass spectrometer. These procedures search for optimum ion production and transmission towards the detector. In addition, the settings for SRM transitions, to be used in quantitative analysis can be optimized in this way. An SRM transition is a combination of precursor ion and product ion and the optimum instrument parameters to measure these ions. Although very useful, the procedures do not account for the important influence of mobile-phase composition and sample or matrix effects on the performance of the electrospray interface.
Solvent Composition Both the solvent composition and interfering contaminants in the sample solution can have distinct influence on the efficiency of ESI for a particular analyte. In general, reversed-phase type mobile phases are applied, ie, mixtures of water and either methanol or acetonitrile containing an acid, base, or buffer. It is often found that most analytes show better response from solutions containing > 50% organic solvent (methanol or acetonitrile in most cases). However, ESI is generally not possible from pure organic solvents. Obviously, the organic content of the mobile phase in LC–MS is more determined by retention characteristics in LC than by ionization and MS detection. It has been demonstrated for a large variety of compounds that a methanol-containing mobile phase leads to a better response than an acetonitrile-containing mobile phase. Post-column and/or sheath-flow addition of high-organic solvents, such as aqueous mixtures with 2-propanol or methoxyethanol, are sometimes applied, especially in the analysis of proteins by ESI-MS.
Electrolyte Additives The composition, pH, and concentration of the buffer used also influences the performance of the ESI interface. Based on the ionization mechanisms, briefly outlined above, one would conclude that an acidic mobile phase is needed for the analysis of an organic base and a basic solution for an organic acid. In this way, the formation of liquid-phase preformed ions is promoted. However, in the ESI-MS analysis of especially small molecules (<1 kDa), the picture appears to be more complex. Wrong-wayaround electrospray has been described, where organic bases are ionized from basic solutions in positive-ion mode without significant loss in response.14 Although most of the current LC–MS interfaces can readily cope with non-volatile mobile-phase additives such as sodium phosphate buffers, they should not be used in routine LC–MS applications. They may lead to more pronounced adduct formation, ie, formation of [M + Na]+ next to [M + H]+, and may lead to significant suppression of the response. Therefore, the use of volatile additives is highly recommended. Mallet and coworkers15 investigated the influence of a number of mobile-phase additives on the response of eight basic drugs in positive-ion mode and eight acidic drugs in negative-ion mode. The influence of formic, acetic, and trifluoroacetic acid, and of ammonium hydroxide, formate, biphosphonate and bicarbonate was investigated as a function of additive concentration. Some of their results are pictured in Fig. 3. The ionization of all compounds, both basic and acidic ones, was found to be suppressed by the addition of strong ion-pairing agents like trifluoroacetic acid or nonafluoropentadecanoic acid. Most compounds respond similarly to the addition of acetic acid and formic acid, although the effect is compound dependent. In positive-ion mode, some compounds
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Fig. 3 Effect of additives, formic acid (FA), trifluoroacetic acid (TFA), ammonium hydroxide (NH4OH), and acetic acid (AcOH) on the relative response of (A) propranolol, and (B) risperidone in electrospray ionization (compared to 50% aqueous methanol, which is 100%). Reproduced from Mallet,C.R.; Lu,Z.; Mazzeo, J.R. Rapid Commun. Mass Spectrom., 2004, 18, 49.
(propranolol, trimethoprim, terfenadine, methoxyverapamil, and reserpine) show ionization enhancement at acid concentrations below 0.5%, while others show ionization suppression (risperidone and benextramine). Somewhat surprisingly, the response of some bases (risperidone, terfenadine, metoxyverapamil, benextramine) are enhanced by addition of ammonium hydroxide. The quaternary ammonium compound pipenzolate is not significantly influenced by additions of ammonium hydroxide or formic or acetic acid. Most basic compounds are suppressed by the addition of ammonium formate, bicarbonate and biphosphonate. In negative-ion mode, the ionization of most compounds is suppressed by formic and acetic acid additives. Ammonium hydroxide results in (significant) ionization enhancement for cholic acid, raffinose, canrenoic acid, while the ionization of fumaric acid, malic acid, and etidronic acid is strongly suppressed. The response of cholic acid, raffinose, and canrenoic acid is also enhanced by ammonium formate and bicarbonate. Ammonium biphosphate suppresses the response of all acidic compounds.
Detergents The influence of various detergents on the electrospray ionization of proteins was also investigated in some detail.16 The detergents are frequently required for the solubilization of the proteins. At concentrations of 0.01% or below, only sodium dodecylsulfate and taurocholate show significant ionization suppression. At 0.1%, most of the detergents result in significant suppression, eg, to below 10% for sodium dodecylsulfate and taurocholate, CTAB, LDAO, and Triton X-100, to the 10–30% range for sodium cholate, and various alkyl glucosides, and to the 30–60% range for CHAPS, hexyl and dodecyl glucosides.
Sample Interferences In quantitative analysis, further ionization suppression or enhancement may be due to co-eluting sample constituents not removed by the sample pre-treatment. This is often indicated as a matrix effect. In most cases, the identity of the components causing the matrix effects is not clear. It has been demonstrated that non-volatile residues in the pre-treated plasma or urine samples and
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especially phospholipids in plasma samples are important causes of matrix effects. If the ionization suppression is not too severe, ie, not compromising the method detection limit too much, the absolute matrix effect would not be of significant importance. However, it is found that a relative matrix effects can occur as well, which are non-reproducible ionization suppression or enhancement effects between different batches of the same sample. Thus, method development for quantitative analysis should include studies on multiple sample batches in order to study relative matrix effects.17,18 To some extent, matrix effects can be overcome by the use of stable-isotope-labelled internal standards (SIL-IS), although results have been reported where SIL-IS and analyte experience different matrix influences. When in method development and validation a matrix effect is observed which compromises the reliability of the method (poor %RSD), further attention must be paid to improving the sample pre-treatment method. The best way to study matrix effects involves the analysis of calibration series of three sets samples: (1) solvent standards containing analyte and SIL-IS, (2) postextraction spiked matrix samples containing analyte and SIL-IS, and (3) pre-extraction spiked matrix samples containing analyte and SIL-IS. Comparison of peak areas (or peak area ratios) between post-extracted spiked samples and solvent standards allows the assessment of the absolute matrix effect. From the peak areas (or peak area ratios) of pre-extraction and post-extraction spiked matrix samples, the extraction recovery can be determined. If samples from a number of independent matrix batches, eg, 5–6 batches, are analysed, the absolute differences of the slopes and the relative standard deviation of the slopes of the calibration plots obtained can be used to assess the relative matrix effects.17,18
Concluding Remarks Being a liquid-phase ionization method, the performance of ESI can be significantly influenced by the solvent composition, especially mobile-phase additives and sample interferences. Nevertheless, ESI-MS can be successfully applied in the (quantitative) analysis of samples from various application areas. In most cases, the method development for LC–MS is easier to perform than for LC–UV. However, one should keep in mind that these two detection techniques respond differently to interfering compounds, and therefore require a different method development and optimization strategies. With proper validation, LC–MS is a powerful approach to quantitative analysis. Care must be taken in the selection of mobile-phase additives and in sample pre-treatment.
Methodologies Based on Electrospray Ionization ESI-MS has become an important tool in analytical chemistry. It is widely applied in LC–MS in many application areas (see below). However, the power of ESI-MS in analyte ionization has also led to a range of derived methods where analyte ionization by ESI is achieved in different experimental setups, not necessarily involving continuous solvent delivery like in LC–MS. The nanoelectrospray interface, discussed above,13 can be applied to couple nano-LC to MS, but also to perform the MS analysis of small samples ( 1 mL) from a nanoelectrospray needle. With flow-rates in the range of 10 nL/min, this allows to perform a range of MS and tandem mass spectrometry (MS–MS) experiments. A different approach to sample analysis is provided by desorption electrospray ionization (DESI).19 In DESI, a high-velocity spray of charged microdroplets from a (pneumatically-assisted) electrospray needle is directed at a surface, mounted in front of the ion-sampling orifice of an API source. DESI releases gas-phase ions from the surface material or surface constituents, which can be analyzed by MS. DESI enables the study of compounds at surfaces without extensive isolation and sample pre-treatment steps, eg, drugs of abuse from tablets, and also opens possibilities to perform chemical imaging of surfaces such as thin-layer chromatography plates and tissue sections.20 In paperspray ionization (PSI), biomolecules are ionized from a paper tip emerged with non-polar solvents like hexane or toluene, placed in the electric field close to the ion-sampling orifice of an API source.21
Applications The advent of ESI-MS certainly opened many new application areas for MS and LC–MS analysis. This is based on the ability to provide extreme soft liquid-based ionization. The most important application area is perhaps the analysis of peptides and proteins. The possibility to perform rapid molecular-weight determination of proteins up to 200 kDa and beyond stimulated the commercial availability of MS instrumentation featuring atmospheric-pressure ion sources, equipped with ESI. Other application areas benefited from these developments. LC–MS has become an important analytical tool in many areas of drug development within the pharmaceutical industry, in the study of natural products in plants and animals, in food and environmental analysis and in the clinical application area for therapeutic drug monitoring, profiling of endogenous compounds for diagnostic purposes, systematic toxicological analysis, and monitoring of inherited metabolic diseases. As indicated, ESI-MS plays an important role in the characterization of proteins. In this area, ESI-MS is complemented by matrixassisted laser desorption ionization MS (MALDI-MS), which in some cases appears to be less prone to ionization suppression effects by sample constituents and/or additives. First of all, the formation of multiple-charge ions of proteins enables the accurate determination of the protein molecular weight, generally within 0.1% accuracy. The ion envelope of multiple-charge proteins ions can actually be a disadvantage, especially in the analysis of proteins with significant heterogeneity due to for instance posttranslational glycosylation. In current protein research, the protein, or even a complete proteome, is often enzymatically digested to
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Fig. 4 Negative ion electrospray ionization mass spectrum of calmodulin (50 mM) in the presence of melittin (50 mM) and 100-mM calcium acetate, pH 5.7. Ions in the range between m/z 900 and 1600 represent uncomplexed calmodulin and ions in the range between m/z 1700 and 3000. represent the noncovalent calmodulin/melittin complex. Reproduced from Veenstra, T.D. et al., J. Am. Soc. Mass Spectrom. 1998, 9, 580–584 with permission from Elsevier Science.
a peptide mixture. The MS characterization of this mixture leads to a peptide map. Due to its ability to produced single-charge ions, MALDI-MS is preferred in most cases in this initial research. Data from the peptide map are applied to bioinformatics tools involving a protein database search in an attempt to identify the protein. If further studies are required to identify the protein, eg, peptide sequencing by means of MS–MS, the use of ESI is often more favorable, because it is coupled more readily to tandemquadrupole, ion-trap, or quadrupole–time-of-flight and orbitrap hybrid instruments. The identification and quantification of proteins in complex biological samples is especially important in the current proteomics research. While for many years MALDI-MS was the method of choice, because of the ease with which it is combined to two-dimensional gel electrophoresis, the role of ESI-MS in combination with multidimensional LC is increasing. Already in the early applications of ESI-MS in the analysis of proteins it was found that ESI can significantly contribute to the study of protein conformation in solution. While initially solvent-induced conformational changes were investigated, such studies were extended by the use of hydrogen/deuterium exchange and other gas-phase reactions. More recently, ion-mobility spectrometry was implemented in MS–MS instruments as a powerful tool in the assessment of conformational changes of proteins. Such tools are not only applied in fundamental studies, but also applied to detection and monitoring of changes in protein conformation related to for instance neurodegenerative diseases like Alzheimer, Parkinson and Creutzfeldt-Jacobs disease. Another important application area of ESI-MS is the study of noncovalent complexes between proteins and inhibitors, cofactors, metal ions, carbohydrates, other peptides and proteins, substrates, and of nucleic acid complexes. In most cases such complexes exhibit relatively low charge states, resulting in high m/z. In most cases, these so-called native-MS studies22 of such complexes requires the use of either modified quadrupole mass analyzers, to enlarge their m/z range, or time-of-flight instrument, which show an inherent larger m/z range. For example, the group of Naylor23 examined the binding of the calcium-binding protein calmodulin to the calmodulin-dependent protein kinase II (CamK-II) and to melittin. Both peptides form equimolar complexes with calmodulin only in the presence of calcium (stoichiometry was 1: 1: 4 for calmodulin: peptide: Ca). A typical spectrum of the calmodulin/melittin complex is shown in Fig. 4. These type of studies are relevant both in the field of structural biology and in the field of drug discovery, enabling the study of drug-protein or inhibitor-protein interactions.
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Blakley, C. R.; Vestal, M. L. Thermospray Interface for Liquid Chromatography/Mass Spectrometry. Anal. Chem. 1983, 55, 750–754. Yamashita, M.; Fenn, J. B. Electrospray ion Source. Another Variation of the Free-jet Theme. J. Phys. Chem. 1984, 88, 4451–4459. Fenn, J. B.; Mann, M.; Men, C. K.; Wong, S. F.; Whitehouse, C. M. Electrospray Ionization — Principles and Practice. Mass Spectrom. Rev. 1990, 9, 37–70. Mann, M.; Meng, C. K.; Fenn, J. B. Interpreting Mass Spectra of Multiply Charged Ions. Anal. Chem. 1989, 61, 1702–1708. Covey, T. R.; Bonner, R. F.; Shushan, B. I.; Henion, J. D. The Determination of Protein, Oligonucleotide and Peptide Molecular Weights by ion-Spray Mass Spectrometry. Rapid Commun. Mass Spectrom. 1988, 2, 249–256. Smith, R. D.; Light-Wahl, K. J. The Observation of non-Covalent Interactions in Solution by Electrospray Ionization Mass Spectrometry: Promise, Pitfalls and Prognosis. Biol. Mass Spectrom. 1993, 22, 493–501. Kebarle, P.; Peschke, M. On the Mechanisms by which the Charged Droplets Produced by Electrospray Lead to gas Phase Ions. Anal. Chim. Acta 2000, 406, 11–35. Niessen, W. M. A. Liquid chromatography-mass spectrometry, 3rd ed.; ISBN 978-0-824-74082-5. Wilm, M. S.; Mann, M. Analytical Properties of the Nanoelectrospray ion Source. Anal. Chem. 1996, 68, 1–8. Zhou, A.; Cook, K. D. Protonation in Electrospray Mass Spectrometry: Wrong-way-Round or Right-way-Round? J. Am. Soc. Mass Spectrom. 2000, 11, 961–966. Mallet, C. R.; Lu, Z.; Mazzeo, J. R. A Study of ion Suppression Effects in Electrospray Ionization from Mobile Phase Additives and Solid-Phase Extracts. Rapid Commun. Mass Spectrom. 2004, 18, 49–58. Ogorzalek Loo, R. R.; Dales, N.; Andrews, P. C. Surfactant Effects on Protein Structure Examined by Electrospray Ionization Mass Spectrometry. Protein Sci. 1994, 3, 1975–1983. Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Strategies for the Assessment of Matrix Effects in Quantitative Bioanalytical Methods Based on HPLC–MS–MS. Anal. Chem. 2003, 75, 3019–3030. Matuszewski, B. K. Standard Line Slopes as a Measure of a Relative Matrix Effect in Quantitative HPLC–MS Bioanalysis. J. Chromatogr. B 2006, 830, 293–300. Taka´ts, Z.; Wiseman, J. M.; Cooks, R. G. Ambient Mass Spectrometry using Desorption Electrospray Ionization (DESI): Instrumentation, Mechanisms and Applications in Forensics, Chemistry, and Biology. J. Mass Spectrom. 2005, 40, 1261–1275. Wu, C.; Dill, A. L.; Eberlin, L. S.; Cooks, R. G.; Ifa, D. R. Mass Spectrometry Imaging Under Ambient Conditions. Mass Spectrom. Rev. 2013, 32, 218–243. Li, A.; Wang, H.; Ouyang, Z.; Cooks, R. G. Paper Spray Ionization of Polar Analytes using Non-Polar Solvents. Chem. Commun. 2011, 47, 2811–2813. Lorenzen, K.; van Duijn, E. Native Mass Spectrometry as a Tool in Structural Biology. Curr. Protoc. Protein Sci. 2010, 62, 17.12.1–17.12.17. Veenstra, T. D.; Tomlinson, A. J.; Benson, L.; Kumar, R.; Naylor, S. Low Temperature Aqueous Electrospray Ionization Mass Spectrometry of Noncovalent Complexes. J. Am. Soc. Mass Spectrom. 1998, 9, 580–584.