Collect. Czech. Chem. Commun. 2000, 65, 1669-1676

Peptide Sequencing by Matrix-Assisted Laser Desorption/Ionisation and Post-Source Decay Mass Spectrometry: A Rapid Method to Design Oligonucleotide Hybridisation Probes for Cloning cDNA Encoding Pyranose 2-Oxidase from Trametes multicolor

Petr Haladaa, Christian Leitnerb, Jindřich Volca, Dietmar Haltrichb and Vladimír Havlíčeka,*

a Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Prague 4, Czech Republic
b Division of Biochemical Engineering, Institute of Food Technology, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria


A rapid and elegant method for designing oligonucleotide hybridisation probes for cloning cDNA encoding a biologically and/or biotechnologically important protein is presented. The approach is based on proteolytic digestion of a protein of interest and subsequent matrix/assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the resulting peptide mixture. The method is demonstrated on the analysis of pyranose 2-oxidase (P2O), a homotetrameric flavoprotein used as a catalyst component in several biotechnologically important carbohydrate conversions. P2O from the fungus Trametes multicolor was cleaved directly in the gel by two different proteases and the peptides formed were subjected to MALDI-MS. A comparison of the obtained peptide maps to those theoretically derived from the known sequence of homologous P2O (Trametes versicolor) allowed us to select peptide candidates for designing hybridisation probes. The suitable peptides were sequenced by post-source decay (PSD) analysis. The acquired sequence data are aimed at cloning and sequencing of T. multicolor p2o cDNA and at production of the recombinant enzyme.

Keywords: Mass spectrometry; Matrix-assisted laser desorption/ionisation; MALDI; Proteins; Sequence; Pyranose 2-oxidase; Oligonucleotide probe; Enzymes.

References: 16 live references.