Amino acid composition and wavelength effects in matrix‐assisted laser desorption/ionization

,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 had the highest proton affinity, underlining their role in MALDI of proteins. Matrix-assisted laser desorption/ionization (MALDI) is effectively used for molecular mass determination of large biopolymers. L* In this method, a pulsed laser is used to irradiate the mixture of sample and matrix. Interaction of the laser beam with the mixture ejects both matrix and analyte molecules into the gas phase. The matrix molecules may undergo extensive fragmentation and ionization. Only a small portion of the biomolecules, however, become ionized and subsequently analyzed by the mass spectrometer. The low ion yield in general, and the variable ionization efficiency in particular, are two important limiting factors in the application of MALDI. Proteins are known to show much stronger response than, for example, carbohydrates or most of the synthetic polymers. Even among similar size proteins there are great differences in the ionization yield. Bovine albumin or cytochrome c, for example, provide ample ion intensities, whereas no analyte specific ions can be observed at the same concentrations of pepsin or collagen. In mixtures of proteins certain components can dominate the spectra independent of relative concentrations. The presence of these components may diminish the response from other proteins bordering on complete suppression of the signal in extreme cases. These severe interferences hamper the applications of MALDI and make quantitation especially challenging. Since the advent of MALDI, there have been several efforts directed toward the understanding of ion formation mechanisms in the laser desorpotion process.3-8