No abstract

Using molecular dynamics simulations we have demonstrated that the guest species in matrix-assisted laser desorption exhibits significantly different conformations in the gas phase, on the crystal surface, and embedded into the matrix. Our model fully accounted for the internal structure of matrix and guest molecules; thus, we were able to follow the details of conformational changes in the guest molecule within the crystal and throughout the desorption. During the embedding process, simulated by sinapinic acid crystal growth around the substance P guest species, changes in the guest species conformation were investigated. While the guest species placed on the surface of the host crystal exhibited two hydrogen bonds on the backbone and one turn, the conformation of the embedded species contained three hydrogen bonds and two turns. Independent of the burial depth, the conformation of the guest species within the crystal remained the same. After laser heating and during the desorption process, no preferred gas-phase conformation was observed. Investigation of the radius-of-gyration of the guest species originating from different layers of the crystal revealed that, upon desorption, the guest species did not sustain a stable conformation in the gas phase. Liftoff velocities of the guest species embedded in various layers of the host crystal and on the surface were similar to the liftoff velocities of the matrix molecules around them. This is an indication of the entrainment of guest species by the matrix plume. Investigation of the energy histories of the matrix and guest species showed lower internal and kinetic temperatures for the latter, which could explain the absence of fragmentation in MALDI. Results also showed that maximum energy transfer occurred during the phase transition of the host crystal at which time the guest species were released from the crystal. Examining the noncovalent complex of the guest species and sinapinic acid anions showed that the complex remained intact throughout the desorption process. A similar phenomenon is often reflected in MALDI mass spectra, in the form of guest-matrix adduct peaks.

Ion yields were investigated in matrix-assisted laser desorption/ionization (MALDI) as a function of amino acid composition using a variable wavelength ion source. In the case of nitrogen laser excitation (337 nm), [M + H]+ ions were abundant for short peptides containing basic or polar amino acid residues. The lack of basic residues led to diminishing ion formation at 337 nm. Increasing the chain length led to enhanced ionization even for peptides with non-polar side chains. In contrast to the liquid phase basicities the basic residues showed Arg > His > Lys order in their affinity to protons. Variations in the average ion yield showed decreasing trend with increasing basicity of the residues pointing to potential differences in the matrix incorporation of different guest molecules. Dye laser excitation at 280 nm resulted in extensive fragmentation and enhanced ion formation from peptides containing aromatic side chains indicating the possible role of analyte excited states in ion formation. Semi-empirical quantum chemical calculations were used to explore the sites of protonation. Heat of formation diagrams of the matrix-analyte complexes were examined as a function of their configuration. Investigation of the optimized geometries allowed the location of bifurcated and linear hydrogen bonds between the non-polar analyte (e.g., Gly-Gly) and matrix molecules. In addition to these intermolecular bonds, in the case of His-His an intramolecular hydrogen bond was also formed within the analyte molecule. Proton affinity values were calculated for every dipeptide while the site of protonation was varied. The results seemed to indicate that for every dipeptide the amino terminus was more susceptible to protonation than the peptide bond. Due to increased stabilization effects in homo-oligomers (Gly,), increasing the number of residues led to an increase in proton affinity. For basic diopeptides (e.g., His-His), the side chains bad the highest proton affinity, underlining their role in MALDI of proteins.

Molecular dynamics was used to analzye energy transfer rates between matrix and guest molecules in matrix-assisted laser desorption allowing for a large number of internal degrees of freedom. The effect of initial matrix temperature jump on internal energy equilibration times and on guest limiting temperatures has been studied on a model system comprising a pyridine-3-carboxylic acid matrix and leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) guest molecule. It appears that the energy transfer rates between matrix and guest molecules depend on the initial matrix temperature jump. The initial matrix temperature of 900 K leads to incomplete desorption, whereas at 1500 K complete desorption and the formation of an energy-transfer bottleneck was observed. Following the guest center-of-mass at different initial matrix temperatures indicates that in the case of 1500 K and 3000 K the desorption process is complete, whereas at 900 K the guest molecule stays near the matrix surface. In the case of complete desorption deeper embedding of the guest molecules leads to somewhat lower guest limiting temperatures. Uniformly higher limiting temperatures are observed for higher laser irradiance and the increase in burial depth leads to more complete equilibration between the two species.

Two nitrogen laser pulses of subthreshold irradiance were used to achieve matrix-assisted laser desorption/ionization in a collinear configuration. Optimum laser pulse delay times yielding maximum guest ion intensities were determined for four different matrices between 4.2 ns for sinapinic acid (SA) and 12.5 ns for 2,5-dihydroxybenzoic acid (DHB). The signal decay times followed an opposite trend to the position of maxima, DHB being the fastest decaying (1.1 ns) and SA the slowest (9.8 ns). Collinear subthreshold experiments allow for a two orders of magnitude improvement in spatial and time resolution over post-ionization experiments.