A high analytical sensitivity in ultraviolet matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is only achieved if the laser wavelength corresponds to a high optical absorption of the matrix. Laser fluence and the physicochemical properties of the compounds, e.g., the proton affinity, also influence analytical sensitivity significantly. In combination, these parameters determine the amount of material ejected per laser pulse and the ion yield, i.e., the fraction of ionized biomolecules. Here, we recorded peptide ion signal intensities as a function of these parameters. Three cinnamic acid matrices were investigated: α-cyano-4-hydroxycinnamic acid, α-cyano-4-chlorocinnamic acid, and α-cyano-2,4-difluorocinnamic acid. In addition, 2,5-dihydroxybenzoic acid was used in comparison experiments. Ion signal intensities "per laser shot" and integrated ion signal intensities were acquired over 900 consecutive laser pulses applied on distinct positions on the dried-droplet sample preparations. With respect to laser wavelength, the two standard MALDI wavelengths of 337/355 nm were investigated. Also, 305 or 320 nm was selected to account for the blue-shifted absorption profiles of the halogenated derivatives. Maximal peptide ion intensities were obtained if the laser wavelength fell within the peak of the absorption profile of the compound and for fluences two to three times the corresponding ion detection threshold. The results indicate ways for improving the analytical sensitivity in MALDI-MS, and in particular for MALDI-MS imaging applications where a limited amount of material is available per irradiated pixel.
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