Thin-Layer Chromatography Combined with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Abstract

The combination of thin-layer chromatography (TLC) with matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been accomplished. The direct coupling protocol includes analyte fraction from inside the TLC plate and its transport to the surface following matrix deposition and crystallization. The fraction procedure was optimized to minimize analyte planar diffusion and to achieve high sensitivitu. Various MALDI matrices were evaluated for use with silica gel and cellulose TLC plates. To avoid a large abundance of cationized peaks, we used TLC plate pretreatment by washing in a methanol/water solution. The best detection limit was obtained with ferutic aid (ferutic acid/fructose) and sinapinic acid matrices. These matrices also showed high sodium and potassium salt tolerance. Use of salts in the developing solvents caused an increase in the cationized peaks without impacting sensitivity. The best crystal homogeneity on silica gel and cellulose as well as surface coverage was obtained using a 2-(4-hydroxyphenylazo)benzoic acid (HABA) matrix. An absolute detection limit of 2-4 ng was demonstrated for bradikin, angiotensin, and enkephalin derivatives. The method also worked for larger peptides and small proteins like bovine insulin chain B, insulin, horse heart cytochrome c, and myoglobin, but TLC separation was poor. Only minor fragmentation was found for all analytes. The influence of ninhydrin on spectral quality was investigated. An increased signal was observed for several analytes when ninhydrin was applied to the TLC plate. The TLC/MALDI method was found to be suitable for direct TLC imaging, which was carried out using the HABA matrix with a spatial resolution of ca. 1 mm. The current protocol induced additional analyte planar diffusion of ∼1.5 mm, but such effects can be eliminated by protocol modification