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Identification of Tuber borchii Vittad. mycelium proteins separated by two‐dimensional polyacrylamide gel electrophoresis using amino acid analysis and sequence tagging
Author(s) -
Vallorani Luciana,
Bernardini Francesca,
Sacconi Cinzia,
Pierleoni Raffaella,
Pieretti Barbara,
Piccoli Giovanni,
Buffalini Michele,
Stocchi Vilberto
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200011)21:17<3710::aid-elps3710>3.0.co;2-9
Subject(s) - isoelectric focusing , chromatography , isoelectric point , chemistry , gel electrophoresis , polyacrylamide gel electrophoresis , edman degradation , molecular mass , immobilized ph gradient , electroblotting , proteome , biochemistry , peptide sequence , gene , enzyme
This paper reports the first results in the proteome analysis of Tuber borchii Vittad. mycelium, an ectomycorrhizal fungus poorly defined genetically, but known for its generation of edible fruit bodies known as white truffles. Employing isoelectric focusing on immobilized pH gradients, followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, we obtained an electropherogram presenting over 800 spots within the window of isoelectric points (p I ) 3.5—9 and a molecular mass of 10—200 kDa. Different reducing agents were tested in the sample preparation buffers, and the standard lysis buffer plus 2% w/v polyvinylpolypyrrolidone allowed the best solubilization and resolution of the proteins. The T. borchii proteins separated in micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and visualized by Coomassie staining. Twenty‐three proteins were excised and analyzed by the combination of amino acid and N ‐terminal analysis. One protein was identified by matching its amino acid composition, estimated isoelectric point and molecular mass against the SWISS‐PROT and EMBL databases. Four spots were successfully tagged by Edman microsequencing but no homologous sequences were found in databases.