Comparative analysis of OFFGel, strong cation exchange with pH gradient, and RP at high pH for first-dimensional separation of peptides from a membrane-enriched protein fraction

Authors

  • Bruno Manadas,

    Corresponding author
    1. Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College of Dublin, Dublin, Ireland
    2. Proteomics Unit, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
    • Center for Neuroscience and Cell Biology, University of Coimbra, Biocant, Parque Tecnológico de Cantanhede, Núcleo 04, Lote 3, 3060-197 Cantanhede, Portugal Fax:+351-231-419-049
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    • These authors contributed equally to this work.

  • Jane A. English,

    1. Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College of Dublin, Dublin, Ireland
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    • These authors contributed equally to this work.

  • Kieran J. Wynne,

    1. Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College of Dublin, Dublin, Ireland
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  • David R. Cotter,

    1. Department of Psychiatry, Royal College of Surgeons in Ireland, RCSI ERC, Smurfit Building, Beaumont Hospital, Dublin, Ireland
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  • Michael J. Dunn

    1. Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College of Dublin, Dublin, Ireland
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Abstract

The analysis and quantitation of membrane proteins have proved challenging for proteomics. Although several approaches have been introduced to complement gel-based analysis of intact proteins, the literature is rather limited in comparing major emerging approaches. Peptide fractionation using IEF (OFFGel), strong cation exchange HPLC using a pH gradient (SCX-pG), and RP HPLC at high pH, have been shown to increase peptide and protein identification over classic MudPIT approaches. This article compares these three approaches for first-dimensional separation of peptides using a detergent phase (Triton X-114) enriched membrane fraction from mouse cortical brain tissue. Results indicate that RP at high pH (pH 10) was superior for the identification of more peptides and proteins in comparison to the OFFGel or the SCX-pG approaches. In addition, gene ontology analysis (GOMiner) revealed that RP at high pH (pH 10) successfully identified an increased number of proteins with “membrane” ontology, further confirming its suitability for membrane protein analysis, in comparison to SCX-pG and OFFGel techniques.

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