Principles of electrospray ionization

Abstract

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.

Fig. 1.

The ion evaporation process. An individual ion leaves the charged droplet in a solvated state. The electric field strength at the surface of a droplet is so high that the energy required to increase the droplet surface is rapidly compensated by the gain because of Coulombic repulsion. k_Reaction, reaction rate constant; k, Boltzmann constant; T, temperature; h, Planck's constant; R, ideal gas constant.

Fig. 2.

The charge residue process. A highly charged droplet shrinks by solvent evaporation until the field strength at the location with the highest surface curvature is so large that a Taylor Cone forms. From the tip of the Taylor Cone, other highly charged smaller droplets are emitted. This process can repeat itself until droplets are formed that contain only one analyte molecule. This molecule is released as an ion by solvent evaporation and declustering. The equation describes the maximum charge a droplet can carry before the Coulomb repulsion overcomes the surface tension. Locally, it is the condition for the formation of a Taylor Cone. q, droplet charge at the Rayleigh instability limit; r, droplet radius; ε₀, electric permittivity of the surrounding medium; γ, surface tension; σ, surface charge density.

Fig. 3.

Typical electrospray mass spectra. (A and B) Show a peptide mixture and, in the detailed view, a triply charged ion recorded with an standard ion trap (A) and an higher resolving orbitrap (B). (C) shows a spectrum of a 47 kDa denatured protein. It is displayed by an entire series of peaks, one for each charge state of the protein. A deconvolution algorithm can construct a spectrum displaying the neutral mass of the protein. In (D), the spectrum of a GroEL chaperonin assembly, an 800 kDa large noncovalent complex, is shown (26). Its high m/z value of 9457 is remarkable. Noncovalent complexes are analyzed under structure conserving conditions and take up only a limited number of charges relative to their large mass.

Fig. 4.

Change of the electrospray ion signal with analyte concentration. A, The standard behavior of the electrospray ion signal with increasing analyte concentration. Over a range of three orders of magnitude, the signal grows linearly with concentration before it saturates. B, The signal dependence of a two component solution. The ion signals start to saturate simultaneously. They level off at the same or at different total ion intensities. If the components differ considerably in hydrophobicity, the more hydrophobic component can even suppress the hydrophilic one at high concentrations (B 3) (16).

Fig. 5.

Conditions for measuring the association constant of a binary protein complex. To use mass spectra as a read out in measuring the association constant of a protein complex with the titration method, at least three conditions have to be fulfilled. The concentrations of the components must remain below the saturation level so that the ion intensities reflect the molecular concentration in solution. The primary droplets have to be small so that all droplets generated evaporate fast and no in-droplet complexes are formed on the basis of higher concentrations. Finally, the desolvation of molecules in the interface region of mass spectrometers has to be gentle so that correctly formed complexes do not dissociate.