Electrospray ionization

FAB and PD have been replaced by electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) in the analytical mass spectrometry laboratory, because both of these newer techniques have a wider mass range of analysis and have lower detection limits. ESI and MALDI have become invaluable ionization techniques for nonvolatile components. This is particularly true for a wide range of biological molecules including proteins, peptides, nucleic acids, etc. Samples can be analyzed by ESI using either direct injection or introduction through liquid chromatography.

Typically ESI forms protonated molecules with little or no fragmentation. High molecular weight molecules can also be ionized using ESI. In this case the molecule becomes multiply-charged and the molecule of interest is observed at its respective m/z. For example, a protein with a molecular weight of 20,000 Da that has 20 protons attached to it, would be detected at m/z 20,020/20, which is equal to 1,001. Figure 5-4 shows the ESI mass spectrum of horse heart myoglobin, with a molecular weight 16,951 Daltons. The M+16H+ and M+15H+ ions result in the peaks at m/z 1060.5 ((16,951 + 16)/16) and 1131.1((16,951 + 15)/15), respectively.

In the case of a protein of unknown molecular weight, the molecular weight can be determined by using equations (4) and (5) and knowing the m/z values of two adjacent multiply-charged ions.

Xi is the mass-to-charge ratio of the ion with the lower m/z, X2 is the mass-to-charge ratio of the ion with the higher m/z, and n is the charge on the ion with the higher m/z. Knowing the charge on a given m/z ion, the molecular weight of the protein can be calculated by using the following equation.

In the example of the horse heart myoglobin, we select X1 = 1060.5 and X2 = 1131.1, n = 15, and MW = 15*1131.1 - 15 = 16,951.5. This is consistent with the known molecular weight of the protein.

1060 5 it3i,i






1211 7



1541 9

1695 9




1500 2000

Figure 5-4. Electrospray ionization mass spectrum of horse heart myoglobin.

The choice of solvents and related components, which must be volatile, is very important for obtaining high-quality electrospray spectra. Even though sample introduction is through a liquid medium, the solvent eventually needs to be volatilized. If a nonvolatile solvent or buffer is used, the result is frequent plugging of the capillary tubing and/or some of the beam-defining components. In ESI the solvated sample is passed through a needle held at a high potential (3-10 kV). As the molecule exits from the needle, the resulting spray undergoes electrostatic nebulization, which places (a) charge(s) on the droplet. The charged droplet passes through a variety of focusing elements, which are differentially pumped. One result is desolvation of the droplet. Depending on the size and chemical makeup of the analyte, the resulting stable ion can have a single charge, or depending on the repulsive forces, may be multiply charged. Mass analysis of this ion can be carried out with any type of mass analyzer, including magnetic sector, quadrupole, ion trap or time-of-flight. Sample concentrations that can routinely be analyzed are sub-picomole/microliter. The detection limit for many components, however, is considerably lower. Table 5-1 gives the results of the ms/ms analysis of a set of betaine analogs. Notice the structurally diagnostic fragment ions available for each of these individual betaines (Wood et al., 2002).

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