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    Comparison of one- and two-dimensional liquid chromatography approaches in the label-free quantitative analysis of methylocella silvestris

    Patel, N.A. and Crombie, A. and Slade, S.E. and Thalassinos, Konstantinos and Hughes, C. and Connolly, J.B. and Langridge, J. and Murrell, J.C. and Scrivens, J.H. (2012) Comparison of one- and two-dimensional liquid chromatography approaches in the label-free quantitative analysis of methylocella silvestris. Journal of Proteome Research 11 (9), pp. 4755-4763. ISSN 1535-3893.

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    Abstract

    The proteome of the bacterium Methylocella silvestris has been characterized using reversed phase ultra high pressure liquid chromatography (UPLC) and two-dimensional reversed phase (high pH)–reversed phase (low pH) UPLC prior to mass spectrometric analysis. Variations in protein expression levels were identified with the aid of label-free quantification in a study of soluble protein extracts from the organism grown using methane, succinate, or propane as a substrate. The number of first dimensional fractionation steps has been varied for 2D analyses, and the impact on data throughput and quality has been demonstrated. Comparisons have been made regarding required experimental considerations including total loading of biological samples required, instrument time, and resulting data file sizes. The data obtained have been evaluated with respect to number of protein identifications, confidence of assignments, sequence coverage, relative levels of proteins, and dynamic range. Good qualitative and quantitative agreement was observed between the different approaches, and the potential benefits and limitations of the reversed phase–reversed phase UPLC technique in label-free analysis are discussed. A preliminary screen of the protein regulation data has also been performed, providing evidence for a possible propane assimilation route.

    Metadata

    Item Type: Article
    Keyword(s) / Subject(s): label-free, reversed phase-reversed phase, methanotroph, quantification, data-independent
    School: Birkbeck Schools and Departments > School of Science > Biological Sciences
    Research Centre: Structural Molecular Biology, Institute of (ISMB)
    Depositing User: Sarah Hall
    Date Deposited: 04 Feb 2014 11:59
    Last Modified: 06 Dec 2016 10:40
    URI: http://eprints.bbk.ac.uk/id/eprint/9160

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