Comparison of gel-based phosphoproteomic approaches to analyse scarce oviductal epithelial cell samples

Authors

  • Birgit Steinberger,

    1. Department for Agrobiotechnology (IFA Tulln), Institute of Biotechnology in Animal Production, University of Natural Resources and Applied Life Sciences Vienna, Tulln, Austria
    2. Institute of Animal Breeding and Genetics, Department for Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria
    Search for more papers by this author
  • Urban Besenfelder,

    1. Reproduction Centre Wieselburg, Department for Biomedical Sciences, Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria
    Search for more papers by this author
  • Gottfried Brem,

    1. Christian Doppler Laboratory for Innovative Immunotherapy, University of Veterinary Medicine, Vienna, Austria
    Search for more papers by this author
  • Corina Mayrhofer

    Corresponding author
    1. Institute of Animal Breeding and Genetics, Department for Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria
    • Department for Agrobiotechnology (IFA Tulln), Institute of Biotechnology in Animal Production, University of Natural Resources and Applied Life Sciences Vienna, Tulln, Austria
    Search for more papers by this author

Correspondence: Dr. Corina Mayrhofer, Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Veterinärplatz 1, 1210 Vienna, Austria

E-mail: corina.mayrhofer@vetmeduni.ac.at

Fax: +43-272-66280-603

Abstract

The reversible change of the phosphorylation state of proteins regulates key cellular processes. In the present study, three different gel-based approaches were compared with regard to their applicability to quantitatively analyse the phosphoproteome of scarce biological material obtained ex vivo. Our results show that the phosphoproteome characterisation of oviductal epithelial cells isolated from the female reproductive tract requires affinity enrichment and pre-electrophoretic labelling using fluorescence dyes. Using this approach, 30 μg of enriched phosphoproteins proved to be sufficient for the phosphoproteome characterisation. In contrast, sequential fluorescence staining of 2D-separated total cell lysates as well as sequential staining in conjunction with a pre-enrichment step led to detection discrepancies and excluded further analysis steps. Information gained from this study provides a successful approach for the phosphoproteome analysis of scarce samples. In addition, the cellular processes taking place in the female reproductive tract can be monitored ex vivo.

Ancillary