• MALDI (matrix-assisted laser desorption/ionization)
• MALDI Matrices: Properties and Requirements
• Sample Preparation Techniques
• Applications and Choice of Matrix
• Literature

MALDI (matrix-assisted laser desorption/ionization),a laser-based soft ionization method has proven to be one of the most successful ionization methods for mass spectrometric analysis and investigation of large molecules.[1,2] In addition analysis of post source decay (decay of proteins following the ionization) in many cases allows rapid sequencing of proteins. Thus MALDI has gained a crucial importance for protein analysis.

Its constituting feature is that the sample is embedded in a chemical matrix (ca. 1000 x molar excess) that greatly facilitates the production of intact gas-phase ions from large, nonvolatile, and thermally labile compounds such as proteins, oligonucleotides, synthetic polymers. and large inorganic compounds. The matrix plays a key role in this technique by absorbing the laser light energy and causing a small part of the target substrate to vaporize.

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Analysis by MALDI mass spectrometry can be divided into two steps.

• The first step involves preparing a sample by mixing the analyte with a molar excess of matrix. The typical matrix for use with ultraviolet lasers is an aromatic acid with a chromophore that strongly absorbs the laser wavelength. Other laser wavelengths are possible, in particular the mid-infrared range where the matrix can be energized by vibrational excitation; different matrix compounds must be used in this case.
• The second step of the MALDI process involves desorption of bulk portions of the solid sample by a short pulse of laser light.

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MALDI Matrices: Properties and Requirements

The MALDI matrix must meet a number of requirements simultaneously:

• be able to embed and isolate analytes (e.g., by co-crystallization)
• be soluble in solvents compatible with analyte
• be vacuum stable
• absorb the laser wavelength
• cause co-desorption of the analyte upon laser irradiation
• promote analyte ionization

It is believed that compounds with labile protons, such as carboxylic acids, are good MALDI matrices in the positive ion mode because they are easily able to protonate neutral analyte molecules in the plume. However, an acidic environment is not always desirable, in particular if denaturation of the tertiary structure of biomolecules should be avoided. Therefore mostly nonacidic matrices are used for protein measurements [3]. Compounds that are not easily protonated can be cationized instead, often by adding a small quantity of salt to the sample (alkali cations, and also Cu or Ag). It is also possible to detect analytes as radical cations by employing so-called electron transfer matrices [4]. More easily deprotonated compounds , such as oligonucleotides, are usually detected in negative ion mode.

The MALDI method has been developed empirically and despite its widespread use, the factors that determine success or failure of MALDI experiments are not yet fully understood. Investigations of the MALDI mechanism have recently become the focus of interest of a number of research groups [5].

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Sample Preparation Techniques

In its current state, MALDI is primarily based on the laser desorption of solid matrix-analyte deposits [6]. The technique suffers from some disadvantages such as low shot-to-shot reproducibility, short sample life time and strong dependence on the sample preparation method, figure 2. A few research groups have investigated the use of liquid matrices [7], to increase sample lifetime and eliminate the search for sweet-spots, by exploiting the self-healing properties of the sampling position through molecular diffusion. Table 1 shows different sample preparation methods.

Table 1: Different sample preparation methods

Solid Matrix	Liquid Matrix	Special preparations
Dried-droplet	Chemical liquid	Solid supports
Vacuum-drying	Particle-doped (two-phase) liquid	MALDI on 2D-gels
Crushed-crystal	Chemical-doped liquid	Insoluble samples
Fast-evaporation	-	-
Overlayer	.	-
Sandwich	-	-
Spin-coating	.	-
Slow-crystallization	-	-
Electrospray	.	-
Quick and dirty	-	-
Matrix precoated targets	.	-

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Applications and Choice of Matrix

The most important applications of MALDI mass spectrometry are (in decreasing order of importance): peptides and proteins, synthetic polymers, oligonucleotides, oligosaccharides, lipids, inorganics. Numerous matrices have been found for these and other classes of compounds; a summary is given in figure 1. Although electrospray ionization (ESI) is somewhat competitive and certainly complementary, MALDI remains the method of choice in several key areas, particularly proteomics. ESI mass spectra include many peaks of multiply charged ions which can complicate the interpretation of the spectra of complex samples. Also, the ion current is distributed over a range of m/z values, sometimes compromising sensitivity. The sensitivity of ESI is also severely reduced by the presence of salts, impurities, and organic buffers which are more easily tolerated by MALDI.

Table 2. Guide to Sample Preparation with FLUKA products and Sigma Calibration Kits