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Type of publication: Journal Article
Type of document: Full Paper

Year: 2013

Authors: Steinberger, B; Besenfelder, U; Brem, G; Mayrhofer, C

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

Source: Proteomics. 2013; 13(1):12-16

Authors Vetmeduni Vienna:

Besenfelder Urban
Brem Gottfried
Itze-Mayrhofer Corina
Steinberger Birgit

Vetmed Research Units
Institute of Animal Breeding and Genetics, Unit of Reproductive Biology

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.

Keywords Pubmed: Electrophoresis, Gel, Two-Dimensional/methods
Epithelial Cells/metabolism
Fallopian Tubes/chemistry
Fallopian Tubes/metabolism
Phosphoproteins*/isolation & purification

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