Separating the wheat from the chaff: Determination of confidently secreted proteins by including information on their cellular abundance.

One important strategy of cells to communicate with their environment is the release of proteins which can serve as signals for other cells nearby or at distant places in a complex organism. A first step in the characterization and investigation of cell-cell communication in this context is to figure out which proteins are released from cells under defined experimental conditions. Here, we present an approach that will give rise to a high-quality secretome and detects proteins that will be confidently released by cultured cells. This approach is based on the separate preparation of proteins from conditioned medium and corresponding cell lysates. After protein digestion and quantitative mass spectrometric analysis, protein abundances are compared and proteins showing a significantly higher abundance in the secretome are identified. We assume that these proteins have a higher probability of being released by well-directed processes and not simply by contamination of (dead) cells. We show an optimized protocol in which samples from primary human normal dermal fibroblasts (NHDF) are prepared with the single-pot solid-phase-enhanced sample preparation (SP3) method from only 450μL conditioned medium along with one-hour gradient separations and data-independent mass spectrometric data acquisition.