title: Assessment of sample preparation methods for metaproteomics of extracellular proteins.
abstract: Enzyme discovery in individual strains of microorganisms is compromised by the limitations of pure culturing. In principle, metaproteomics allows for fractionation and study of different parts of the protein complement but has hitherto mainly been used to identify intracellular proteins. However, the extracellular environment is also expected to comprise a wealth of information regarding important proteins. An absolute requirement for metaproteomic studies of protein expression, and irrespective of downstream methods for analysis, is that sample preparation methods provide clean, concentrated and representative samples of the protein complement. A battery of methods for concentration, extraction, precipitation and resolubilization of proteins in the extracellular environment of a constructed microbial community was assessed by means of 2D gel electrophoresis and image analysis to elucidate whether it is possible to make the extracellular protein complement available for metaproteomic analysis. Most methods failed to provide pure samples and therefore negatively influenced protein gel migration and gel background clarity. However, one direct precipitation method (TCA-DOC/acetone) and one extraction/precipitation method (phenol/methanol) provided complementary high quality 2D gels that allowed for high spot detection ability and thereby also spot detection of less abundant extracellular proteins.
authors.affiliation: Molecular Biotechnology, Dept. of Physics, Chemistry and Biology, Linköping University, SE-581 83 Linköping, Sweden.
authors.forename: Jutta
authors.initials: J
authors.lastname: Speda
authors.affiliation: Molecular Biotechnology, Dept. of Physics, Chemistry and Biology, Linköping University, SE-581 83 Linköping, Sweden.
authors.forename: Mikaela A
authors.initials: MA
authors.lastname: Johansson
authors.affiliation: Molecular Biotechnology, Dept. of Physics, Chemistry and Biology, Linköping University, SE-581 83 Linköping, Sweden.
authors.forename: Uno
authors.initials: U
authors.lastname: Carlsson
authors.affiliation: Molecular Biotechnology, Dept. of Physics, Chemistry and Biology, Linköping University, SE-581 83 Linköping, Sweden; InZymes Biotech AB, Gjuterigatan 1B, SE-582 73 Linköping, Sweden. Electronic address: marka@ifm.liu.se.
authors.forename: Martin
authors.initials: M
authors.lastname: Karlsson
mesh_terms: D001419:Bacteria / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D040901:Proteomics / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D001426:Bacterial Proteins
keywords: 2D gel electrophoresis; Enzyme discovery; Extracellular; Metaproteome; Microbial community; Sample preparation