Ordered porous alumina geometries and surface metals for surface-assisted laser desorption/ionization of biomolecules: Possible mechanistic implications of metal surface melting

Platinum- or gold-coated porous alumina with submicrometer structures is a potential substrate for surface-assisted laser desorption/ionization (SALDI) mass spectrometry of biomolecules, not requiring sample matrixes. In this study, a highly ordered porous alumina substrate was fabricated to study the geometric factors allowing good SALDI performance. Evaluation was based on the signal-to-noise ratio of protonated angiotensin I ions in the mass spectrum obtained by 337-nm ultraviolet laser irradiation. Varying the geometries, including pore densities and diameters, revealed the laser intensity required to generate ions to be related to surface porosity. Surface platinum was melted upon laser irradiation at the fluence sufficient to generate peptide ions as confirmed by scanning electron microscopy. Moreover, a thin (5-20 nm) platinum coat requires a low intensity of laser light for desorption/ionization. Considering the size effect on the melting of metals, our findings suggest the surface platinum melting to be involved in ion generation from this SALDI substrate type. Indeed, tantalum, which has a higher melting point, required more laser fluence to generate ions. The porous alumina layer beneath surface metals probably worked as a thermal insulator. This double-layer-type substrate allowed ionization of angiotensin I and verapamil at low femtomole levels. Moreover, small proteins and glycoproteins such as 24-kDa trypsinogen and 15-kDa ribonuclease B could be ionized with sufficient sensitivity on this target. Taking advantage of matrix-free methods, concentrating the sample solution in the target concavity or widening the laser beam focus enhanced the signal-to-noise ratio for analyte ions in the mass spectrum. Activity is maintained for months in air.