Matrix-assisted Laser Desorption/Ionization in Transmission Geometry: Instrumental Implementation and Mechanistic Implications

Abstract

Matrix-assisted laser desorption/ionization (MALDI) spectra of peptides and proteins in the mass range 1–150 kDa, obtained with the same instrument using both the usual top illumination and also using a novel transmission illumination geometry are reported. 2,5-Dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid were used as matrices. In transmission geometry, ions are generated by MALDI rather than laser-induced pressure pulses, they originate from the irradiated sample face. Spectra obtained with the two geometries are virtually indistinguishable with only minor differences in threshold fluence, signal-to-noise ratio, mass resolution and number of successful exposures from a given sample. Spectra were also obtained from specially mounted cytochrome c-doped single crystals of DHB. Using delayed ion extraction, the initial velocity of ions upon extraction was measured. In the transmission geometry, the mean initial velocities were zero for delay times of >0.5 μs, in contrast to a mean axial velocity of ca. 650 m/s under prompt extraction conditions. This result is interpreted as a randomization of the directions of the initial velocity distribution, resulting in a partial or full thermalization of the desorption plume in the confined space between the crystal bottom face and the glass substrate. A mathematical analysis of the time-of-flight equation for ions from such thermalized MALDI-plumes is presented.