Detection of Neutral Species in the MALDI Plume Using Femtosecond Laser Ionization: Quantitative Analysis of MALDI-MS Signals Based on a Semiequilibrium Proton Transfer Model

We investigated neutral species in the matrix-assisted laser desorption and ionization (MALDI) plume using femtosecond laser ionization spectrometry with simultaneous measurement of the standard MALDI spectrum of the identical MALDI event induced by pulsed UV laser irradiation. The ratio of neutral species in the plume [A]_p/[M]_p (A = phenylalanine (Phe) or alanine (Ala), M = 2,5-dihydroxybenzoic acid (DHB)) was confirmed to be the same as that of the sample mixture in the range of [A]₀/[M]₀ = 4 × 10^-4-1, indicating the validity of the widely adopted approximation [A]_p/[M]_p = [A]₀/[M]₀ in the reaction quotient of the proton transfer reaction MH⁺ + A ⇄ M + AH⁺. An effective parameter representing the extent of thermal equilibrium in the thermal proton transfer model is introduced for the first time. Numerical simulation based on this semiequilibrium model successfully reproduced variations of MALDI signal intensities AH⁺ and MH⁺ with two parameters: the fraction of ionized matrix a ≤ 10^-5 and an effective temperature T = 1200 and 1100 K for Phe/DHB and Ala/DHB systems, respectively. These values show good agreement with those determined previously by different experimental approaches. The extent of thermal equilibrium was determined to be 95% and 98% for Phe/DHB and Ala/DHB systems, respectively, suggesting that the proton transfer reactions almost proceed to their thermal equilibrium.