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

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Title: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
Creators:
Halada, Petr
Leitner, Christian
Volc, Jindřich
Haltrich, Dietmar
Havlíček, Vladimír
Journal or Publication Title:
Collection of Czechoslovak Chemical Communications, 65, 10, pp. 1669-1676
Uncontrolled Keywords:Enzymes, Mass spectrometry, Oligonucleotide probe, Proteins, Matrix-assisted laser desorption/ionisation, Pyranose 2-oxidase, MALDI, Sequence

Abstract

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 <i>Trametes multicolor</i> 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 (<i>Trametes versicolor</i>) 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 <i>T. multicolor p2o</i> cDNA and at production of the recombinant enzyme. <p>

Title: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
Creators:
Halada, Petr
Leitner, Christian
Volc, Jindřich
Haltrich, Dietmar
Havlíček, Vladimír
Uncontrolled Keywords:Enzymes, Mass spectrometry, Oligonucleotide probe, Proteins, Matrix-assisted laser desorption/ionisation, Pyranose 2-oxidase, MALDI, Sequence
Divisions:Life and Chemical Sciences > Institute of Organic Chemistry and Biochemistry > Collection of Czechoslovak Chemical Communications
Journal or Publication Title:Collection of Czechoslovak Chemical Communications
Volume:65
Number:10
Page Range:pp. 1669-1676
ISSN:0010-0765
E-ISSN:1212-6950
Publisher:Institute of Organic Chemistry and Biochemistry
Related URLs:
URLURL Type
http://dx.doi.org/10.1135/cccc20001669UNSPECIFIED
ID Code:1748
Item Type:Article
Deposited On:06 Feb 2009 17:12
Last Modified:06 Feb 2009 16:12

Citation

Halada, Petr; Leitner, Christian; Volc, Jindřich; Haltrich, Dietmar; Havlíček, Vladimír (2000) 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. Collection of Czechoslovak Chemical Communications, 65 (10). pp. 1669-1676. ISSN 0010-0765

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