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The incorporation of analytes into matrix crystals and even more so its mechanistic aspects as a prerequisite for a successful MALDI-MS has been discussed controversially in the literature. Solventless sample preparation techniques can shed new light on this question. In order to investigate some crucial aspects of these preparation techniques, lyophylized peptides and proteins were ground or milled with the powder of two different matrices, 2,5-DHB as incorporating matrix and 2,6-DHB for which protein incorporation was definitely excluded in a prior study, and pressed into pellets. The dependence of the quality of the UV-MALDI-spectra on the mass (up to 12,360 Da) and the milling time in a ball mill is reported. For mellitin different initial axial ion velocities were found, when desorbed from 2,5-DHB-pellets as prepared and after wetting and re-drying. Velocities of 150 and 580 m s−1 for dry and wetted pellets are taken as representative for hard desorption from a surface and soft desorption of matrix-incorporated analytes, respectively. Proteins labeled with either fluorescein isothiocyanate (FITC) or Texas Red (TR) were nano-electrosprayed onto a bed of ferulic acid in a ‘dry’ or ‘wet’ mode. All ‘dry’ deposits exhibit strong fluorescence but do not yield MALDI-ion signals. All ‘wet’ deposits yield MALDI-signals of the proteins; the fluorescence of FITC is quenched in ‘wet’ deposits because of the low matrix pH.

In this study, high resolution field emission scanning electron microscopy (FE-SEM) was tested for its feasibility to investigate analyte incorporation into and analyte distribution in slowly grown crystals of 2,5-dihydroxybenzoic acid (2,5-DHB) and 2,6-dihydroxybenzoic acid (2,6-DHB); both compounds function as a matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). To investigate matrix–analyte interaction in 2,5-DHB crystals two Au-labels (20 nm colloidal gold and Nanogold) were examined as potential protein markers to visualize proteins in matrix solids by SEM. For this purpose, analyte-doped 2,5-DHB crystals were mechanically cleaved and SEM-micrographs of the opened inner faces were recorded. During the course of the Au-label study, crystal defects became apparent for crystals grown from analyte-free and analyte-doped matrix solutions; these defects are interpreted as ‘fluid’ or ‘liquid inclusions.’ The size and amount of liquid inclusions depend on the individual cooling rate during crystal growth and on the size of analyte molecules added to the matrix solution prior to crystal growth. Considering the results obtained for both matrix compounds, we assume that analyte incorporation occurs via a ‘solid solution’ in the case of 2,5-DHB as earlier proposed and not via phase defects such as liquid inclusions. Whether liquid inclusions—presumably filled with saturated mother solution—help to facilitate the desorption/ionization event remains open.

Since the early days of matrix-assisted laser desorption/ionization (MALDI), the definition of a unified theory on the MALDI process is a major challenge. The new results presented in this paper clearly show that the idea of a uniform MALDI mechanism that accounts for the spreading applications has to be given up. Based on different preparation protocols distinct differences in the desorption/ionization process for carbohydrates in contrast to peptides/proteins are elucidated. Although isolated/incorporated and cluster-desorbed peptides/proteins are effectively entrained and cooled within the expanding plume of matrix clusters, as shown by a low degree of metastable analyte-ion fragmentation, a laser desorption and gas-phase cationization mechanism can be confirmed as the dominant part in ionization for neutral oligosaccharides which can be initiated even for particulate analyte material or deposits onto a matrix surface. The previously presented “lucky survivor-model” on cluster desorption of preformed ions thus needs this extension.

The 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-positional isomers of dihydroxybenzoic (DHB) acid were investigated with respect to their function as matrix-assisted laser desorption/ionization (MALDI) matrices. Optical absorption spectra of solid matrix samples, recorded in diffuse reflection from samples revealed peak broadening and a red shift of the peaks relative to the solution spectra. Single crystals of all isomers were grown from solution and analyzed by x-ray crystallography. Single crystals, as well as standard dried droplet and thin layer preparations with added cytochrome c, were analyzed by UV- (266, 308, 337, 355 nm) and IR- (2.94 μm) MALDI time-of-flight mass spectrometry (MALDI-TOF-MS). A spectrophotometric measurement of the heme absorption around 400 nm of redissolved single crystals showed a quantitative incorporation of the protein into crystals of 2,5-DHB and a partial protein incorporation with large statistical fluctuations into single crystals of 2,4-DHB. No protein incorporation above the detection limit of 10−5 molar analyte-to-matrix ratio was found for the other isomers. Best MALDI spectra from standard preparations were recorded for dried droplet preparations of 2,5-DHB at 337 and 355 nm and for 2,4-DHB at 266 and 308 nm. Both matrices performed well in the IR, too. 2,6-DHB yielded spectra of comparable quality at 337 nm and 2.94 μm, but only when prepared in a thin layer from an acetone solution. The results suggest that protein incorporation into the crystals of solid MALDI matrices is helpful, but not a prerequisite for MALDI. A large surface-to-volume ratio, typical for microcrystalline thin layer preparations supports protein desorption if no measurable incorporation occurs. Undesirable matrix adduct formation to the protein ions was seen for all DHB isomers at the wavelengths of 308 and 266 nm.