title: A novel proteomics sample preparation method for secretome analysis of Hypocrea jecorina growing on insoluble substrates.
abstract: Analysis of the secretomes of filamentous fungi growing on insoluble lignocellulosic substrates is of major current interest because of the industrial potential of secreted fungal enzymes. Importantly, such studies can help identifying key enzymes from a large arsenal of bioinformatically detected candidates in fungal genomes. We describe a simple, plate-based method to analyze the secretome of Hypocrea jecorina growing on insoluble substrates that allows harsh sample preparation methods promoting desorption, and subsequent identification, of substrate-bound proteins, while minimizing contamination with non-secreted proteins from leaking or lysed cells. The validity of the method was demonstrated by comparative secretome analysis of wild-type H.jecorina strain QM6a growing on bagasse, birch wood, spruce wood or pure cellulose, using label-fee quantification. The proteomic data thus obtained were consistent with existing data from transcriptomics and proteomics studies and revealed clear differences in the responses to complex lignocellulosic substrates and the response to pure cellulose. This easy method is likely to be generally applicable to filamentous fungi and to other microorganisms growing on insoluble substrates.
authors.affiliation: Norwegian University of Life Sciences, NMBU, Department of Chemistry, Biotechnology and Food Science, P.O. Box 5003, NO-1432 Aas, Norway. Electronic address: oskar.bengtsson@nmbu.no.
authors.forename: Oskar
authors.initials: O
authors.lastname: Bengtsson
authors.affiliation: Norwegian University of Life Sciences, NMBU, Department of Chemistry, Biotechnology and Food Science, P.O. Box 5003, NO-1432 Aas, Norway.
authors.forename: Magnus Ø
authors.initials: MØ
authors.lastname: Arntzen
authors.affiliation: Norwegian University of Life Sciences, NMBU, Department of Chemistry, Biotechnology and Food Science, P.O. Box 5003, NO-1432 Aas, Norway.
authors.forename: Geir
authors.initials: G
authors.lastname: Mathiesen
authors.affiliation: Norwegian University of Life Sciences, NMBU, Department of Chemistry, Biotechnology and Food Science, P.O. Box 5003, NO-1432 Aas, Norway.
authors.forename: Morten
authors.initials: M
authors.lastname: Skaugen
authors.affiliation: Norwegian University of Life Sciences, NMBU, Department of Chemistry, Biotechnology and Food Science, P.O. Box 5003, NO-1432 Aas, Norway.
authors.forename: Vincent G H
authors.initials: VGH
authors.lastname: Eijsink
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D049109:Cell Proliferation / Q000502:physiology; D002474:Cell-Free System / Q000378:metabolism*; D035901:Hypocrea / Q000378:metabolism*; D008031:Lignin / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D012995:Solubility; D013048:Specimen Handling / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D020543:Proteome; C036909:lignocellulose; D008031:Lignin
keywords: Filamentous fungi; Quantitative proteomics; Sample preparation; Secretome; Solid substrate