期刊
ANALYTICAL BIOCHEMISTRY
卷 516, 期 -, 页码 23-36出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2016.10.008
关键词
Enzyme discovery; Microbial community; Metaproteome; Extracellular; Sample preparation; 2D gel electrophoresis
资金
- Swedish Research Council [621-2009-4150]
- Tekniska Verken i Linkoping AB
- InZymes Biotech AB
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 extra cellular 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. (C) 2016 Elsevier Inc. All rights reserved.
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