Principles of Electrospray Ionization

Electrospray ionization is today the most widely used ionization technique in chemical and biochemical analysis. Interfaced with a mass spectrometer it allows the investigation of the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized. There is no limitation in mass which thus enables even the investigation of large noncovalent protein complexes. Its high ionization efficiency profoundly changed biomolecular sciences because proteins can be identified and quantified on trace amounts in a high throughput fashion. This review article focuses mainly on the exploration of the underlying ionization mechanism. Some ionization characteristics are discussed that are related to this mechanism. Typical spectra of peptides, proteins, and noncovalent complexes are shown and the quantitative character of spectra is highlighted. Finally the possibilities and limitations in measuring the association constant of bivalent noncovalent complexes are described. Electrospray ionization is today the most widely used ionization technique in chemical and biochemical analysis. Interfaced with a mass spectrometer it allows the investigation of the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized. There is no limitation in mass which thus enables even the investigation of large noncovalent protein complexes. Its high ionization efficiency profoundly changed biomolecular sciences because proteins can be identified and quantified on trace amounts in a high throughput fashion. This review article focuses mainly on the exploration of the underlying ionization mechanism. Some ionization characteristics are discussed that are related to this mechanism. Typical spectra of peptides, proteins, and noncovalent complexes are shown and the quantitative character of spectra is highlighted. Finally the possibilities and limitations in measuring the association constant of bivalent noncovalent complexes are described. With the discovery in the late 1980s of two soft ionization techniques, electrospray and matrix assisted laser desorption/ionization (MALDI) 1The abbreviations used is:MALDImatrix assisted laser desorption/ionization., a very important and long-lasting limitation of mass spectrometers as analytical instruments was removed—the restriction in the molecular weight of the analytes. With MALDI and electrospray, molecules with masses beyond 1000 Da could be transferred into the gas phase and ionized with very high efficiency and without any obvious limitation in mass (1Karas M. Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons.Anal. Chem. 1988; 60: 2299-2301Crossref PubMed Scopus (4837) Google Scholar, 2Meng C.K. Mann M. Fenn J. Of protons or proteins.Zeitschrift für Physik D Atoms, Molecules and Clusters. 1988; 10: 361-368Crossref Scopus (267) Google Scholar, 3Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules.Science. 1989; 246: 64-71Crossref PubMed Scopus (6335) Google Scholar). It was not the first time that molecules with masses in the range of 10,000 Da were seen in a mass spectrometer, but the enormous transfer and ionization efficiency of these two methods opened entirely new areas of research in chemistry, biochemistry, and biology. matrix assisted laser desorption/ionization. Electrospray, as a method to dissipate a liquid sample in a homogeneous form, is an old technique. Its underlying physical effect was first described by Sir Geoffrey Taylor (4Taylor G. Disintegration of water drops in an electric field.Proc. R. Soc. Lon. A. 1964; 280: 383-397Crossref Google Scholar). Very early, it was speculated by Malcolm Dole et al. that electrospray could be used to generate molecular beams of large molecules, but he could not underpin this speculation with convincing experiments; his experimental setup was too limited (5Dole M. Mack L.L. Hines R.L. Mobley R.C. Ferguson L.D. Alice M.B. Molecular beams of macroions.J. Chem. Phys. 1968; 49: 2240-2249Crossref Scopus (1160) Google Scholar). Only in 1988 could John Fenn's group demonstrate that it was possible to transfer large molecules, such as proteins, as ions into the gas phase without breaking them apart (2Meng C.K. Mann M. Fenn J. Of protons or proteins.Zeitschrift für Physik D Atoms, Molecules and Clusters. 1988; 10: 361-368Crossref Scopus (267) Google Scholar, 3Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules.Science. 1989; 246: 64-71Crossref PubMed Scopus (6335) Google Scholar). Other research groups and companies already working with electrospray or similar spray techniques interfaced to mass spectrometers took up the discoveries rapidly, which was the beginning of the now broad use of mass spectrometers in biomolecular sciences. Today, electrospray is the most widely used technique for the analysis of samples in liquid form. Because it ionizes molecules directly from the liquid phase, it is compatible with traditional chromatographic separation techniques widely used in analytical chemistry. Equally important is that it is the most universally known ionization method with very low chemical specificity. Ions released by electrospray are very stable and not in an excited state, which can lead to their rapid decay like many ions generated by MALDI. The ionization process is unlimited in mass (6Siuzdak G. Bothner B. Yeager M. Brugidou C. Fauquet C.M. Hoey K. Chang C.M. Mass spectrometry and viral analysis.Chem. Biol. 1996; 3: 45-48Abstract Full Text PDF PubMed Scopus (149) Google Scholar). These characteristics, paired with its very high ionization efficiency, are the basis of the wide distribution of electrospray ion sources (7Wilm M. Mann M. Analytical properties of the nanoelectrospray ion source.Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1711) Google Scholar). The main focus of this article is to review the mechanistic description of the electrospray ionization process. Research into the ionization process must develop a deeper understanding of how the ions are generated with the ultimate aim of a mathematical description. Such an understanding is important for the optimal design of electrospray ion sources and their interfaces to mass spectrometers. Thus, ionization characteristics like the generated charge state and the transfer of molecular complexes into the gas phase can be manipulated. The central question to answer is as follows: how can ions be generated from charged liquid droplets? Immediately after the discovery that an electrospray ion source can generate large molecular ion beams, two models for the ionization process were proposed: the ion evaporation model (8Iribarne J. Thompson B. On the evaporation of small ions from charged droplets.J. Chem. Phys. 1976; 64: 2287-2294Crossref Scopus (950) Google Scholar, 9Thompson B. Iribarne J. Field induced ion evaporation from liquid surfaces at atmospheric pressure.J. Chem. Phys. 1979; 71: 4451-4463Crossref Scopus (485) Google Scholar) and the charge residue model (5Dole M. Mack L.L. Hines R.L. Mobley R.C. Ferguson L.D. Alice M.B. Molecular beams of macroions.J. Chem. Phys. 1968; 49: 2240-2249Crossref Scopus (1160) Google Scholar, 10Wilm M.S. Mann M. Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last?.Int. J. Mass Spectrom. Ion Proc. 1994; 136: 167-180Crossref Scopus (875) Google Scholar). More recent research refers to these two models and gathers data to find a mathematical description to support either one or the other. In this evaluation process, it should be understood that a model remains a model; it never describes the reality in an absolute way. It is a simplification. A good model should explain experimental data and should have a good predictive power for experimental results in a qualitative or even quantitative way. A model is not true because it is believed to be true. It is even difficult to say that certain experimental findings confirm the model description. It is more accurate to say that the experimental data can be interpreted within the framework of a specific model. The ion evaporation model was originally developed by Iribarne and Thomson to explain the generation of atomic ions from randomly charged droplets produced by a spray atomizer (8Iribarne J. Thompson B. On the evaporation of small ions from charged droplets.J. Chem. Phys. 1976; 64: 2287-2294Crossref Scopus (950) Google Scholar, 9Thompson B. Iribarne J. Field induced ion evaporation from liquid surfaces at atmospheric pressure.J. Chem. Phys. 1979; 71: 4451-4463Crossref Scopus (485) Google Scholar). Droplets shrink by evaporation until the field strength at their surface is sufficiently large that solvated ions can be expelled from the droplet (see Fig. 1). The energy gain in the strong electric field at the surface of the droplet compensates for the required energy to enlarge the surface of the droplet very rapidly when the solvated ion is expelled. There are three important characteristics of this process. The first is a geometric parameter: ion evaporation is expected to play a role when the droplet reaches a very small size of about 20 nm in diameter. Second, the reaction rate kinetic is heavily influenced by chemical properties of the ion. The ion evaporation rate constant depends exponentially on the difference in reaction free enthalpy that needs to be overcome when the ion is expelled from the droplet (see Fig. 1). Even though the ion is solvated by a small shell, this ΔG is a function of the physicochemical properties of the ion itself. Iribarne and Thomson realized that their model cannot explain the evaporation rates of different ions in their experiments. Ions with very different solvation energies had similar reaction rate constants, and simultaneously, other ions with very similar solvation energies had very different evaporation rates (9Thompson B. Iribarne J. Field induced ion evaporation from liquid surfaces at atmospheric pressure.J. Chem. Phys. 1979; 71: 4451-4463Crossref Scopus (485) Google Scholar). Finally, at the onset of ion evaporation, the surface charge density of the droplet is below the maximal possible surface charge density at the Rayleigh limit (see below). In their early work, Fenn and coworkers favored the evaporation model over the charge residue model as an for the generation of large ions from droplets J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules.Science. 1989; 246: 64-71Crossref PubMed Scopus (6335) Google Scholar). The charge residue model that the electrospray process droplets that one analytical ion (see Fig. The ion is released when the A that for a of a droplet with a of nm on one characteristics of this model are that the ionization rate is of the ion. The generation of very small droplets and the efficiency of evaporation the ion and not the physicochemical properties of the ion. The ionization process is not limited by the mass of the complexes can be expected to the process because are by evaporation and not have to overcome an energy with in an electric The charge to the depends on the Rayleigh limit because the droplet from a process by a Taylor The of the ionization process is the electrospray of a The process is It is for the of the ionization mechanism. a is to a liquid in a the liquid is into an The is in such a that is two for of its A surface to the liquid into the to the surface The the liquid to the The is at a certain the surface its to a the very of the a spray is Sir Geoffrey Taylor that an of surface and can be for of its surface for a liquid with an of (4Taylor G. Disintegration of water drops in an electric field.Proc. R. Soc. Lon. A. 1964; 280: 383-397Crossref Google Scholar). the is the the two is for a specific at the of the M.S. Mann M. Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last?.Int. J. Mass Spectrom. Ion Proc. 1994; 136: 167-180Crossref Scopus (875) Google Scholar). the for the that at a specific the Taylor the of the the can which is the when the Taylor and the electrospray is M.S. Mann M. Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last?.Int. J. Mass Spectrom. Ion Proc. 1994; 136: 167-180Crossref Scopus (875) Google Scholar). The Taylor description is a description and not A charged surface with an a for the electric the liquid to This description the generated droplets are charged to their the Rayleigh limit J. R. J. evaporation and in electrospray Phys. Chem. A. Scopus Google Scholar). The charge density at the the that the electric field the surface for a this is the of the Rayleigh the droplets are their to are in molecules the droplet as to an in the field density at the surface of the droplets R.L. and of charged droplets generated by electrospray Phys. Chem. A. PubMed Scopus Google Scholar). In a the field density is and a new Taylor on the which charged small droplets R.L. and of charged droplets generated by electrospray Phys. Chem. A. PubMed Scopus Google Scholar, A. K. and of droplets in 1994; Scopus Google Scholar, J. electrospray mass Chem. 1994; Scopus Google Scholar). the droplet is not this process at an of the which is the with the the electric field density on the surface is the The Rayleigh is not for the which is droplets Taylor at a below the Rayleigh limit for the The the droplets and the the surface the more the droplet be and the the to the Rayleigh limit A. K. and of droplets in 1994; Scopus Google Scholar). This process on droplets and on droplets because their charge density is already to the Rayleigh limit when are The of droplet is about of the of the Thus, a of very small droplets is which are most the source of ions by a mass the very small droplets are the source of ions by a mass spectrometer is by an of electrospray The of a spray of droplets by this in the mass spectrometer results in the ion In the Taylor process can lead to a very of liquid without evaporation of the The of many droplets with on the of nm or to be The source was to generate this of droplet as droplets and is one of the most electrospray with of up to (7Wilm M. Mann M. Analytical properties of the nanoelectrospray ion source.Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1711) Google Scholar, 10Wilm M.S. Mann M. Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last?.Int. J. Mass Spectrom. Ion Proc. 1994; 136: 167-180Crossref Scopus (875) Google Scholar). Ion evaporation as an ionization mechanism. The high of atomic ions generated by liquid ion sources are by an ion evaporation ion and Phys. Scopus Google Scholar). In a liquid ion a high is to a liquid in a until a Taylor The electric field at its high that ions directly of the liquid into the can be used to a very and ion The question is ion evaporation is for electrospray generated ions atmospheric in it is the for the of large molecular the very of the of ion evaporation is the for the reaction rate constant (see Fig. 1). Ion should on the molecular solvation energy to an Even the generated ion be in a state, which the different of should be small this could not be (9Thompson B. Iribarne J. Field induced ion evaporation from liquid surfaces at atmospheric pressure.J. Chem. Phys. 1979; 71: 4451-4463Crossref Scopus (485) Google Scholar, of ion in electrospray mass spectrometry on the of the in the Chem. Scopus Google Scholar). for molecular molecules have a ionization efficiency molecules can even the ion of of ion in electrospray mass spectrometry on the of the in the Chem. Scopus Google Scholar). It should be that is an for this effect within the charge residue model (see below). Ion evaporation is a for Taylor of charged it it to in at a surface charge density that is below the Rayleigh Ions of a should be charged droplets of the size at this most proteins a charge that to the charge of a droplet of the size at its Rayleigh limit Electrospray ionization of large charged Dole's charged residue Scopus Google Scholar). The charge residue model can explain many of the electrospray ionization of large the Taylor it is possible to generate charged droplets that are small that on one M.S. Mann M. Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last?.Int. J. Mass Spectrom. Ion Proc. 1994; 136: 167-180Crossref Scopus (875) Google Scholar). The unlimited mass and the on noncovalent complexes are a of the process (6Siuzdak G. Bothner B. Yeager M. Brugidou C. Fauquet C.M. Hoey K. Chang C.M. Mass spectrometry and viral analysis.Chem. Biol. 1996; 3: 45-48Abstract Full Text PDF PubMed Scopus (149) Google Scholar). The of the ionization of chemical properties of the is because the process depends more on the of the spray and the evaporation characteristics of the The high of the large ions generated with electrospray in to other ionization techniques is by the evaporation process R. Ion energy the charge residue model for small ion by spray Mass Spectrom. PubMed Scopus Google Scholar). of ions charge distribution from the surface of the droplet to the charge on the of the most in the about the electrospray ionization process is the one by that proteins are most charged up to the Rayleigh limit of droplets of the size Electrospray ionization of large charged Dole's charged residue Scopus Google Scholar). This is a strong that their state is a droplet generated the Taylor mechanism. There are two that to with the charge residue the ionization rate for molecules and the of the electrospray ionization process for of the these can be within the framework of the charge residue model. electrospray ion sources at rates of or rate is high that their droplets are in of a certain evaporation these droplets a Taylor droplet process. These droplets are about one of the droplets and a charge already to their Rayleigh these droplets a Taylor process, which the droplets that one and to the molecular ions a droplet one of Taylor droplet it of its charge but about of its mass J. R. J. evaporation and in electrospray Phys. Chem. A. Scopus Google Scholar, A. K. and of droplets in 1994; Scopus Google Scholar). the spray most of the mass of droplets from the surface of Only a small of the mass of droplets up in droplets that to surface molecules have a of in a mass molecules electrospray ionization to character of small Chem. PubMed Scopus Google Scholar). In when a of the liquid is in such a that molecules can be The the charge residue model is the for In a sample with a can a high of without the by it R. M. a electrospray ionization Soc. Mass Spectrom. 10: PubMed Scopus Google Scholar). when a with the ion is the ion of ions in the Some are but are not the Only when the to the for ions more R. M. a electrospray ionization Soc. 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The of nm droplets in a strong field was The model that ion evaporation and of even can about solvated ions are from the droplet by and droplet after In the for the electrospray ionization process is that molecular say 1000 are generated from one analytical ion to the charge residue model. ions can be from field evaporation in a solvated state, as described by the ion evaporation model. 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The is the analysis of entirely into the by a and analysis of mass spectrometry of The is to the of that Only a assisted analysis a deeper of the of on the molecular Electrospray interfaced mass spectrometers are the to the molecular