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Membrane proteomics by high performance liquid chromatography–tandem mass spectrometry: Analytical approaches and challenges

Authors

  • Dajana Vuckovic,

    1. Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada
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    • These authors contributed equally to this work.

  • Laura F. Dagley,

    1. Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada
    2. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
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    • These authors contributed equally to this work.

  • Anthony W. Purcell,

    1. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
    2. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
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  • Andrew Emili

    Corresponding author
    1. Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada
    • Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada
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Correspondence: Dr. Andrew Emili, Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1

E-mail: andrew.emili@utoronto.ca

Fax: +1 416 978 7437

Abstract

Membrane proteins (MPs) play diverse biologically important structural and functional roles including molecular transport, cell communication, and signal transduction. The dysfunctions of many are linked to deleterious human diseases and thus are of utmost importance in drug discovery. MPs comprise approximately 20–30% of all open reading frames (ORFs), however they are typically under-represented in many LC-MS proteomics experiments due to their low abundance and poor solubility. To address these analytical challenges, various MP enrichment, solubilization, digestion, and fractionation strategies have been employed to further improve the coverage of the membrane systems while maintaining compatibility with MS detection. This review discusses both established and emerging high-throughput gel-free analytical workflows in membrane proteomics, and the inherent advantages, disadvantages, and orthogonality of the various approaches. The issues of critical importance for successful LC-MS/MS detection such as detergent selection and minimizing ion suppression in detergent-based workflows are discussed in detail. Recent studies comparing the performance of different analytical strategies are highlighted in order to provide practical insight into the choice of the most appropriate method for membrane-centric applications ranging from cell surface biomarker discovery to MP interaction network mapping.

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