Sophisticated strategies to analyze cell surface proteins are indispensable to study fundamental biological processes, such as the response of cells to environmental changes or cell-cell communication. Herein, we describe a refined mass spectrometry-based approach for the specific characterization and quantitation of cell surface proteins expressed in the female reproductive tract. The strategy is based on in situ biotinylation of rabbit oviducts, affinity enrichment of surface exposed biotin tagged proteins and dimethyl labeling of the obtained tryptic peptides followed by LC-MS/MS analysis. This approach proved to be sensitive enough to analyze small sample amounts (<1μg) and allowed further to trace the dynamic composition of the surface proteome of the oviductal epithelium in response to male gametes. The relative protein expression ratios of 175 proteins were quantified. Thirty-one of them were found to be altered over time, namely immediately, 1h and 2h after insemination compared to the time-matched control groups. Functional analysis demonstrated that structural reorganization of the oviductal epithelial cell surface was involved in the early response of the female organ to semen. In summary, this study outlines a workflow that is capable to monitor alterations in the female oviduct that are related to key reproductive processes in vivo.
Biological significance: The proper interaction between the female reproductive tract, in particular, the oviduct and the male gametes, is fundamental to fertilization and embryonic development under physiological conditions. Thereby the oviductal epithelial cell surface proteins play an important role. Besides their direct interaction with male gametes, these molecules participate in signal transduction and, thus, are involved in the mandatory cellular response of the oviductal epithelium. In this study we present a refined LC-MS/MS based workflow that is capable to quantitatively analyze the expression of oviductal epithelial cell surface proteins in response to insemination in vivo. A special focus was on the very early interaction between the female organ and the male gametes. At first, this study clearly revealed an immediate response of the surface proteome to semen, which was modulated over time. The described methodology can be applied for studies of further distinct biological events in the oviduct and therefore contribute to a deeper insight into the formation of new life.
Keywords: Cell surface proteins; Dimethyl labeling; LC–MS/MS; Oviduct epithelial cells; Quantitation; Semen.
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