title: Comparison of gel-based phosphoproteomic approaches to analyse scarce oviductal epithelial cell samples.
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.
authors.affiliation: Department for Agrobiotechnology (IFA Tulln), Institute of Biotechnology in Animal Production, University of Natural Resources and Applied Life Sciences Vienna, Tulln, Austria.
authors.forename: Birgit
authors.initials: B
authors.lastname: Steinberger
authors.forename: Urban
authors.initials: U
authors.lastname: Besenfelder
authors.forename: Gottfried
authors.initials: G
authors.lastname: Brem
authors.forename: Corina
authors.initials: C
authors.lastname: Mayrhofer
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004847:Epithelial Cells / Q000378:metabolism; D005187:Fallopian Tubes / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006801:Humans; D010750:Phosphoproteins / Q000145:classification / Q000302:isolation & purification; D010766:Phosphorylation; D040901:Proteomics
chemical_list: D010750:Phosphoproteins