2.4- Dihydroxy-6- -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.

Figure 14.6 Structures of some commonly used MALDI matrices (a) 2,5-dihydroxy benzoic acid (DHB), (b) a-cyano-4-hydroxy-cinnamic acid, and (c) sinapinic acid (SA). Combination of these acidic MALDI matrices with organic bases leads to the formation of ionic liquid matrices.

More recently benzoic acid derivatives (MW 122-170) have also been shown to be astringent (17). The most astringent compounds, salicylic (2-hydroxy benzoic acid) and gentisic (2,5 dihydroxy benzoic acid) acids, were ortho substituted, but neither had vicinal hydroxyl groups. Both derivatives had lower pHs than the non-ortho substituted ones, which may have contributed not only to sourness but also to astringency. McManus et al (1981) (18) proposed previously that simple phenols which contain 1,2 dihydroxy or 1,2,3 trihydroxy groups (such as epicatechin or catechin) may cross link and thereby precipitate proteins. It could be speculated that ortho substitution conveys some kind of binding capability similar to that of flavan-3-ols or polyphenolics of hi er MW. 

Refs 4 6) 208-10° (Ref 7) 5-Nitro-2.4-dihydroxybenzoic Acid, yel pits or ndls + H20 (from w), mp 215°(dec) forms several colored cryst salts (Ref 1) 3-Nitro-2,5-dihydroxybenzoic Acid, yel ndls (from benz or w), mp 158° (Ref 2) 4-Nitro-2,5-dihydroxy-benzoic Acid, brn crysts (from w), mp 242°, bp - sublimes w/o decompn (Refs 4 6) 4-Nitro-3,5-dihydroxybenzoic Acid, dk-red ndls (from w), mp - dec 238-40° (Ref 2)... 

Wl Raney nickel is the least active of all of these catalysts with the possible exception of the W8 variety. This inactivity has been used to advantage, for instance, in the hydrogenation of 2,5-dihydroxy benzoic acid to cyclohexane carboxylic acid-3,5-dione (Eqn. 12.10). 2 jj,e use of the more active W7 catalyst for this reaction, resulted in considerable over-reduction of the product. 

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. 

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