Reproducibility of Temperature-Selected Mass Spectra in Matrix-Assisted Laser Desorption Ionization of Peptides

Matrix-assisted laser desorption ionization of peptides was investigated using α-cyano-4-hydroxycinnamic acid as the matrix. In each experiment, a set of mass spectra was collected by repetitive irradiation of a spot on a sample. Even though shot-to-shot variation in spectral pattern was significant, it was reproducible for different spots and samples. Each spectrum was tagged with the temperature in the early plume (<i>T</i>_early) estimated through kinetic analysis of the peptide ion survival probability. <i>T</i>_early decreased as the shot continued because the thermal conduction got more efficient as the sample got thinner. From each spectral set collected under various experimental conditions, a spectrum tagged with a particular <i>T</i>_early was selected. Then, patterns of the spectra thus selected were the same. The reaction quotient for the matrix-to-peptide proton transfer determined at a specified <i>T</i>_early was independent of the sample composition, indicating quasi-thermal equilibrium for this reaction. Furthermore, the van’t Hoff plots were linear, also indicating quasi-thermal equilibrium. This, together with the thermal kinetics for the fragmentation of peptide and matrix ions, is responsible for the reproducibility of the mass spectral pattern at a specified <i>T</i><sub>early