RESEARCH ARTICLE Ahmed Abbas Æ Hasan Koc Æ Feng Liu Æ Ming Tien Fungal degradation of wood: initial proteomic analysis of extracellular proteins of Phanerochaete chrysosporium grown on oak substrate Received: 28 July 2004 / Revised: 12 October 2004 / Accepted: 20 October 2004 / Published online: 18 November 2004 Ó Springer-Verlag 2004 Abstract Two-dimensional (2-D) gel electrophoresis was used to separate the extracellular proteins produced by the white-rot fungus Phanerochaete chrysosporium. So- lid-substrate cultures grown on red oak wood chips yielded extracellular protein preparations which were not suitable for 2-D gel analysis. However, pre-washing the wood chips with water helped decrease the amount of brown material which caused smearing on the acidic side of the isoelectric focusing gel. The 2-D gels from these wood-grown cultures revealed more than 45 protein spots. These spots were subjected to in-gel digestion with trypsin followed by either peptide fingerprint analysis by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or by liquid chromatography (LC)/MS/MS sequencing. Data from both methods were analyzed by Protein Prospector and the local P. chrysosporium annotated database. MALDI- TOF/MS only identified two proteins out of 25 analyzed. This was most likely due to problems associated with glycosylation. Protein sequencing by LC/MS/MS of the same 25 proteins resulted in identification of 16 proteins. Most of the proteins identified act on either cellulose or hemicellulose or their hydrolysis products. Thus far no lignin peroxidase, Mn peroxidase or laccases have been detected. Keywords White-rot fungi Æ 2-D gel Æ MALDI-TOF Æ Peptide mapping Introduction Cellulose, hemicellulose and lignin accounts for the majority of the carbons fixed by photosynthesis (Kirk 1983). The degradation of these components plays a major role in carbon recycling on earth. While cellulose and hemicellulose are degraded by a large number of microbes, lignin is highly recalcitrant and is degraded predominantly by fungi (Crawford and Crawford 1980). Of these fungi, the most extensively characterized are the white-rot fungi (Kirk 1983). White-rot fungi degrade both the lignin and carbohydrate components of woody biomass (Kirk and Farrell 1987). This complicated process involves an ensemble of both oxidative enzymes and hydrolytic enzymes. Cellulose and hemicellulose are degraded by hydrolytic enzymes whereas lignin, a much more formidable substrate, is degraded by oxidative enzymes (Kirk and Cullen 1998). In 1998, Kirk and Cullen (1998) wrote ‘‘The picture of how the hyphae bring about the decay of wood is becoming increasingly clear at the molecular level, even though much remains to be learned about the specific enzymes of the white-rot fungi per se.’’ Although many of the hydrolytic and oxidative enzymes have been purified and their cDNAs isolated, little is known about their expression on woody substrates. The recent sequencing of the genome of the white-rot fungus Phanerochaete chry- sosporium (Martinez et al. 2004) now allows for proteo- mic methods to identify all of the enzymes involved in the degradation of woody substrates. Furthermore, this approach can potentially lead to the discovery of new enzymes involved in wood decay and also provide information on the succession of enzymes involved in this process. Here, advances in instrumentation such as ma- trix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and capillary liquid chromatography–nanoelectrospray ionization– tandem MS (CapLC-ESI-MS/MS), coupled with the very old method of two-dimensional (2-D) gel electrophoresis, now allow proteins of small samples sizes to be identified Communicated by U. Ku¨ck A. Abbas Æ F. Liu Æ M. Tien (&) Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA E-mail: mxt3@psu.edu Tel.: +1-814-8631165 Fax: +1-814-8638616 H. Koc Proteomics and Mass Spectrometry Core Facility, Pennsylvania State University, University Park, PA 16802, USA Curr Genet (2005) 47: 49–56 DOI 10.1007/s00294-004-0550-4