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    J Proteome Res. 2007 Jan;6(1):298-305.

    The proteomic reactor facilitates the analysis of affinity-purified proteins by mass spectrometry: application for identifying ubiquitinated proteins in human cells.

    Source

    Ottawa Institute of Systems Biology, 451 Smyth Road, Ottawa, Ontario, K1H 8M5, Canada.

    Abstract

    Mass spectrometry (MS) coupled to affinity purification is a powerful approach for identifying protein-protein interactions and for mapping post-translational modifications. Prior to MS analysis, affinity-purified proteins are typically separated by gel electrophoresis, visualized with a protein stain, excised, and subjected to in-gel digestion. An inherent limitation of this series of steps is the loss of protein sample that occurs during gel processing. Although methods employing in-solution digestion have been reported, they generally suffer from poor reaction kinetics. In the present study, we demonstrate an application of a microfluidic processing device, termed the Proteomic Reactor, for enzymatic digestion of affinity-purified proteins for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Use of the Proteomic Reactor enabled the identification of numerous ubiquitinated proteins in a human cell line expressing reduced amounts of the ubiquitin-dependent chaperone, valosin-containing protein (VCP). The Proteomic Reactor is a novel technology that facilitates the analysis of affinity-purified proteins and has the potential to aid future biological studies.

    PMID:
    17203973
    [PubMed - indexed for MEDLINE]

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