Identification of shed proteins from Chinese hamster ovary cells: Application of statistical confidence using human and mouse protein databases

The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation to develop a fundamental understanding of the bystander response. Chinese hamster ovary cells were chosen because they have been widely used for radiation studies and are reported to respond to radiation by releasing factors into the media that cause genomic instability and cytotoxicity in unexposed cells, i.e., a bystander effect. Media samples taken for irradiated cells were evaluated using a combination of tandem- and Fourier transform-ion cyclotron resonance (FT-ICR)-mass spectrometry (MS) analyses. Since the hamster genome has not been sequenced, MS data was searched against the mouse and human protein databases. Nearly 150 proteins identified by tandem mass spectrometry were confirmed by FT-ICR. When both types of MS data were evaluated, using a new confidence scoring tool based on discriminant analyses, about 500 proteins were identified. Approximately 20% of these identifications were either integral membrane proteins or membrane associated proteins, suggesting that they were derived from the cell surface and, hence were likely shed. However, estimates of quantitative changes, based on two independent MS approaches, did not identify any protein abundance changes attributable to the bystander effect. Results from this study demonstrate the feasibility of global evaluation of shed proteins using MS in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool.