Matrix Dihydroxy benzoic acid

A lot of features of MALDI are conveyed by its name it is a desorption ionization, produced by a laser beam, and assisted by a matrix (Figure 2.5). The analyte (1 pmol or less) is mixed with a suitable matrix in a 1 1000 or higher molar ratio. The matrix is composed of a compound with a strong absorption at the wavelength of the laser used. These two factors, matrix excess and its strong absorption, ensure that the energy from the laser pulse is absorbed by the matrix and not by the analyte, thus avoiding its decomposition. Nicotinic acid, sinapinic acid (SA), 2,5-dihydroxy benzoic acid (2,5-DHB) and 2-(4-hydroxyphenylazo)benzoic acid (HABA) are some of the most commonly used matrices for MALDI. 

Fig. 10.1. MALDI-TOF mass spectra of (a) porcine cytochrome C from 2,5-dihydroxy-benzoic acid matrix at 337 nm and (b) a monoclonal antibody from nicotinic acid matrix at 266 nm. Reproduced fromRef. [15] by permission. John Wiley Sons, 1991.

Sections, 10-12 p thick, were cut from flash-frozen tissue, thaw-mounted on MALDI plates, and dried in a desiccator prior to deposition of the matrix. Sublimation of 2,5-dihydroxy-benzoic acid over -4 min resulted in a homogenous 5-10 p coating. Alternatively, the tissue was dry-coated with matrix that was passed through a 20 p sieve for 20 min. Both methods were stable in the vacuum for 24 h, although back-sublimation is a risk that can create artificial analyte concentration gradients. 

The samples were prepared by mixing the lignin water soluhons with acetone (4 mg/ml, 50/50 water/acetone by volume). The sample soluhons so prepared were mixed with an acetone soluhon of the matrix (10 mg matrix soluhon per ml acetone). As the matrix 2,5-dihydroxy benzoic acid was used. For enhancement of ion formahon, NaCl was added to the matrix (10 mg/ml in water). The soluhons of the sample and the matrix were mixed in proportions 3 parts matrix soluhon 4- 3 parts lignin soluhon -I- 1 part NaCl soluhon, and 0.5 to 1 pi of the resulhng soluhon mix were placed on the MALDI target. After evaporation of the solvent the MALDI target was introduced into the spectrometer. The dry droplet sample preparahon method was used. 

FIGURE 10.9 MALDI-TOF mass spectra of soybean trypsin inhibitor blotted onto PVDF membrane, using (a) UV laser at 355 nm and (b) IR laser at 2.94 p. Matrix is 2,5-dihydroxy benzoic acid in ethanol. Reprinted with permission from reference 15. 

For MALDI-MS the analyte as a pure compound or a mixture is co-crystallized with a matrix that absorbs laser radiation and promotes ionization. Matrix materials ideal for peptides and proteins are aromatic acids such as sinapinic acid, 2,4-dihydroxy-benzoic acid, and a-cyano-4-hydroxycinnamic acid, each having slightly different ionization characteristics. Published protocols for optimization of sample preparation try to achieve multiple, evenly distributed, small crystals. It is often necessary to remove salts by washing the crystals with water after they have been deposited. 

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