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Matrix-assisted Laser Desorption/Ionisation

Lasers were first utilised in ion sources during the late 1960’s and early 1970’s. It was quickly found that laser light, incident on a pure solid or liquid sample deposited on a sample slide, could be used to create small inorganic and organic gas-phase ions.[1] It was possible to avoid thermal decomposition of fragile analyte molecules by carefully controlling the power density of the laser beam.

The upper mass for laser desorption of biological molecules appears to be about 1000 Da, and for synthetic polymers can be up to a few thousand. Higher-mass ions, however, require higher laser fluences and are destroyed by the laser light. The use of a matrix, together with the analyte, was introduced by Tanaka and co-workers to facilitate desorption/ionisation of high-mass molecules without inducing fragmentation.[2] The breakthrough came from the novel sample preparation that was used. The analyte was dissolved in glycerol, and this glycerol solution was mixed with a fine cobalt powder. The glycerol is virtually transparent at 337 nm, the wavelength of the laser beam utilised, but the cobalt particles would have absorbed the light with high efficiency. The cobalt particles heated up severely, and heat was transferred to the surrounding glycerol/analyte solution. Energy was transferred to the analyte molecules desorbing them into the gas-phase, where they were detected as ions. Experiments using lysozyme as the analyte yielded singly and doubly charged ions.

In 1985 Hillenkamp and co-workers promoted the hypothesis that mixing an analyte with an organic matrix, chosen to absorb at the wavelength of the laser, could ionise large molecules.[3, 4] It was expected that the ablated matrix would absorb sufficient light to result in its ablation, carrying some of the analyte material with it into the gas phase. In the dense cloud above the sample surface, chemical reactions would occur giving a charge to the analyte molecules, and allowing their detection by time-of-flight mass spectrometry.

The first experimental demonstration utilised nicotinic acid as the matrix material for the analysis of large polypeptides.[5] An aqueous solution of the analyte biomolecule was mixed with an aqueous solution of the nicotinic acid, deposited onto a stainless steel substrate, and dried. The ratio of matrix-to-analyte molecules was approximately 1000:1. A Nd:YAG laser was used to irradiate the crystallised mixture at 266 nm, the wavelength at which the nicotinic acid was strongly absorbing. Adjustment of the laser power and matrix-to-analyte ratio for each of the analytes studied gave high signal-to-noise mass spectra.

The advantage of the Hillenkamp method of preparation over the one introduced by Tanaka is the sensitivity, which is especially important for the analysis of precious biological materials. The Hillenkamp method routinely gives high quality results using femtomoles of analyte, while the Tanaka method requireed nanomoles. The Hillenkamp method, known as matrix-assisted laser desorption/ionisation has been demonstrated to be extraordinarily efficient for the desorption and ionisation of many different types of large molecule, including proteins,[6, 7, 8, 9] carbohydrates[10, 11] and synthetic polymers.[12, 13, 14]

The mechanism of ion formation in MALDI is not well understood, and is currently a matter of active research. An understanding of ionisation pathways should help to maximise ion yields, control fragmentation and give access to new classes of analyte. It is clear that only a few types of ion are commonly observed in MALDI. These are radical cations, protonated molecules and cationised molecules in the form of metal-ion adducts. Many and varied chemical and physical pathways have been suggested for MALDI ion formation, including gas-phase photoionisation, ion-molecule reactions, disproportionation, excited-state proton transfer, energy pooling, thermal ionisation and desorption of pre-formed ions. An excellent review of ‘ion formation in MALDI mass spectrometry’ has been given by Zenobi and Knochenmuss.[15]

Matrices in Matrix-assisted Laser Desorption/Ionisation

The selection of a compound for use as a MALDI matrix can be particularly difficult. A suitable MALDI matrix must, firstly, be soluble in solution with the analyte. If this is not possible the matrix and analyte must be deposited separately onto the sample stage in layers.

A second necessary characteristic is to have a strong absorption coefficient at the wavelength of the laser. It is usually easy to determine the absorption coefficient of a solution of the compound, but it is the solid-phase values which are relevant as they are often red-shifted with respect to the solution values. Research has concentrated on the near-UV region, limiting the choice of matrices to aromatic compounds with electron-withdrawing groups.

A further requirement for a matrix is to be chemically inert in terms of reactivity with the analyte.
Potential matrix compounds must also have low sublimation rates, as the sample must be introduced into vacuum for mass spectrometric analysis. High sublimation rates would result in significant changes in the matrix-to-analyte ratio during an experiment, affecting the results.

Even when these criteria are met, most potential matrices do not yield ions when experimentally tested. Nicotinic acid and 2,5-dihydroxybenzoic acid were introduced as matrix materials by Karas and Hillenkamp.[6, 16] Beavis and Chait introduced several derivatives of cinnamic acid, such as 3-methoxy-4-hydroxycinnamic acid and 3,5-dimethoxy-4-hydroxycinnamic acid,[17] and alpha-cyano-4-hydroxycinnamic acid.,[18]

Commonly used MALDI Matrices:


1) Johnston, P. M.; Bergman, M. R.; Zakheim, D., J. Chem. Phys., 1975, 62, 2500
2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T., Rapid Commun. Mass Spectrom., 1988, 2, 151
3) Karas, M.; Bachmann, D.; Hillenkamp, F., Anal. Chem., 1985, 57, 2935
4) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F., Int. J. Mass Spectrom. Ion Proc., 1987, 78, 53
5) Karas, M.; Hillenkamp, F., Anal. Chem., 1988, 60, 2299
6) Hillenkamp, F.; Karas, M., Methods in Enzymlogy, 1990, 193, 280
7) Spengler, B., J. Mass Spectrom., 1997, 32, 1019
8) Mo, W.; Takao, T.; Shimonishi, Y., Rapid Commun. Mass Spectrom., 1997, 11, 1829
9) Ross, P. L.; Davis, P. A.; Belgrader, P., Anal. Chem., 1998, 70, 2067
10) Garozzo, D.; Nasello, V.; Spina, E.; Sturiale, L., Rapid. Commun. Mass Spectrom., 1997, 11, 1561
11) Spengler, B.; Kirsch, D.; Lemoine, J.; Kaufmann, R., J. Mass Spectrom., 1995, 30, 782
12) Smith, P. B.; Pasztor Jr., A. J.; McKelvy, M. L.; Meunier, D. M.; Froelicher, S. W.; Wang, F. C.-Y., Anal. Chem., 1997, 69, 95R
13) O’Connor, P. B.; Duursma, M. C.; van Rooij, G. J.; Heeren, R. M. A.; Boon, J. J., Anal. Chem., 1997, 69, 2751
14) Whittal, R. M.; Schriemer, D. C.; Li, L., Anal. Chem., 1997, 69, 2734
15) Zenobi, R.; Knochenmuss, R., Mass Spectrom. Rev., 1998, 17, 337-366
16) Strupat, K.; Karas, M.; Hillenkamp, F., Int. J. Mass Spectrom. Ion Proc., 1991, 111, 89
17) Beavis, R. C.; Chait, B. T., Rapid Commun. Mass Spectrom., 1989, 3, 432
18) Beavis, R. C.; Chaudhary, T.; Chait, B. T., Org. Mass Spectrom., 1992, 27, 156

All of the above text is an excerpt from:

Bottrill, A.R., Ph.D. Thesis, University of Warwick, 2000.
'High-energy Collision-induced Dissociation of Macromolecules using Tandem Double-focusing/Time-of-flight Mass Spectrometry.'

maintained by:
Dr. Gerhard Saalbach