To understand how a MALDI-TOF instrument works, it is easiest to start with the MALDI source (Fig. 2A). The sample to be analyzed is mixed with a chemical matrix, which typically contains a small organic molecule with a desirable chromophore that absorbs light at a specific wavelength. Typical matrix compounds include 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), and a-cyano-4-hydroxycinnamic acid. The admixture of sample and matrix is then spotted onto a small plate or slide and then allowed to evaporate in air. The evaporation of residual water or other solvent from the sample allows the formation of a crystal lattice into which the peptide sample is integrated. The target is then placed into the source. The source is equipped with a laser, which fires a beam of light at the target. The matrix chemicals absorb photons from the beam and become electronically excited. This excess energy is then transferred to the peptides or proteins in the sample, which are then ejected from the target surface into the gas phase.
Fig. 2. Schematic representation of a MALDI-TOF mass spectrometer. (A) The MALDI ionization process. (B) A MALDI-TOF instrument operating in linear mode. (C) A MALDI-TOF instrument equipped with a reflectron.
This ionization process produces both positive and negative ions, depending on the nature of the sample. For peptides and proteins, the positive ions are almost always the species of interest. The positive ions are formed by accepting a proton as they are ejected from the matrix. Each peptide molecule tends to pick up a single proton. Thus, most of the resulting peptide ions are singly charged. For a peptide of mass 1032, the addition of a proton and its one positive charge makes the m/z value 1033 for the [M+H]+ ion. The ions formed in the MALDI source are then extracted and directed into the TOF mass analyzer.
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