MALDI and Related Methods: A Solved Problem or Still a Mystery?

Abstract

MALDI ionization mechanisms remain a topic of controversy. Some of the major modern models are compared, with emphasis on those of the author. Primary formation, secondary reaction, and loss mechanisms are considered.

Keywords: MALDI; ablation; primary and secondary ionization; recombination.

Fig. 1. Molecular dynamics simulation of a MALDI event showing total density (a), temperature (b) and charge density (c). At each time point (vertical axis), laterally averaged properties are shown. The laser is incident from the right. To the left, outside the image, the solid matrix material continues. In (a), the darkest gray corresponds to the cold solid, the lightest to single molecules per pixel. In (c) the highest charge density (bright yellow) was over 150 per pixel, but rapidly decays to single charges. Evident are the widely varying speeds of the different layers, expansion cooling and temperature gradients, and the large difference between early and late charge densities.

Fig. 2. Rate equation calculations of MALDI ion intensities for DHB matrix and two peptidic analytes, with molecular weights 900 and 1000. Panel (a) shows positive ion spectra; panel (b) negative. The matrix-analyte charge transfer reaction free energies were: analyte 1: −150 kJ/mol (positive) and −50 kJ/mol (negative). Analyte 2: −75 kJ/mol in both polarities. The matrix : analyte mole ratios in the sample before ablation are indicated for each spectrum. Both analytes were present in equal amounts. Between M/A=1000 and 200, the matrix suppression effect (MSE) becomes apparent. At higher analyte concentrations, the analyte suppression effect (ASE) occurs. These effects have also been demonstrated experimentally, as noted in the text.

Fig. 3. Rate equation calculations of MALDI ion intensity ratios for the analytes of Fig. 2, versus time during the MALDI event. The upper curve is the ratio of positive analyte 1 to positive analyte 2 ions, the lower curve is the corresponding negative ion ratio. The matrix/analyte mole ratio in the sample was 0.005 for both analytes. Since the analyte 1 charge transfer reaction with matrix is 100 kJ/mol more favorable than that of analyte 2, a large positive ratio is expected, if no kinetic limitations exist. However, the ratio is modest, 1.05. The opposite should be true in negative mode, analyte 2 is 25 kJ/mol more favorable than analyte 1. Again the ratio deviates by less than 15% from 1. However, in neither case does the ratio correspond to the mole ratios in the sample. Note also the pronounced time dependence, which is a consequence of the multiple coupled formation, transfer and loss mechanisms.

Fig. 4. MALDI ion ratios for some ionic liquids, versus concentration in the sample before ablation, adapted from ref. . The liquids with the higher binding energies, and therefore higher ion–ion recombination energies, give larger signals, at all concentrations. This is contrary to the usual expectation that reactions with larger driving force are faster. On the other hand, it is consistent with a tunneling model for recombination reactions, in the Marcus inverted region. The analytes were BC: benzyltriphenylphosphonium chloride, BP: 1-butyl-3-methylimidazolium hexafluorophosphate, and TB: trihexyltetradecylphosphonium bis(2,4,4-trimethylpentyl)phosphinate.

Fig. 5. Molecular dynamics simulation of a backside MALDI ablation event, showing time-dependent density. The laser is incident from the left. The most energized, highly charged and thoroughly vaporized material is trapped for a significant time behind cold, thick layers. This can be compared to the very rapid escape of top-layer ions and the decreased time for secondary reactions in Fig. 1a.