Matrix-assisted laser desorption/ionization of protein samples containing a denaturant at high concentration using a mid-infrared free-electron laser (MIR-FEL)

Abstract

A novel matrix-assisted laser desorption/ionization (MALDI) method was developed for insoluble protein analysis. Solubilizing agents, such as 8 M urea, used for preparation of insoluble proteins do not absorb typical desorption lasers and impede transient laser heating in the conventional MALDI process. A mid-infrared free-electron laser (MIR-FEL) can individually activate various chemicals by tuning the MIR-FEL wavelength to selectively excite a vibrational mode. In the present method designated UV/FEL-MALDI, the same position on a sample deposit containing a denaturant at high concentration is exposed to a nitrogen laser pulse which is absorbed by UV-matrix and a MIR-FEL macropulse which is absorbed by denaturant in the same time frame. This scheme lets a denaturant at high concentration to be used for the MALDI sample preparation of insoluble proteins. The simultaneous irradiation of a nitrogen laser and MIR-FEL achieves spatially and temporally defined desorption, which is essential to TOFMS detection, while specificity and selectivity owing to the MIR-FEL wavelength can be conserved. The ability of UV/FEL-MALDI to detect the analyte which is deeply embedded in denaturant was examined using model protein samples dissolved into 8 M urea solution. In comparison to the conventional UV-MALDI, orders-of-magnitude improvement in signal-to-noise ratio was obtained by UV/FEL-MALDI with the MIR-FEL wavelength tuned around the absorption maximum of urea. The intact protein of human hair keratin, which is extremely insoluble and is not amenable to UV-MALDI and any other ionization methods, was subjected to UV/FEL-MALDI-TOFMS analysis. The molecule-related cluster ions of γ-keratin were detected for the first time.