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 in an effort to develop a fundamental understanding of the bystander response. CHO cells were chosen for this study because they have been widely used for radiation studies and since they have been 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 FTICR-mass spectrometry analysis. Since the hamster genome has not been sequenced, mass spectrometry data was searched against the mouse and human proteins databases. Nearly 150 proteins that were identified by tandem mass spectrometry were confirmed by FTICR. When both types of mass spectrometry data were evaluated with a new confidence scoring tool, which is based on discriminant analyses, about 500 protein 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, hence were likely shed. However, estimates of quantitative changes, based on two independent mass spectrometry 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 mass spectrometry in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool.
- Research Organization:
- Pacific Northwest National Lab., Richland, WA (US)
- Sponsoring Organization:
- US Department of Energy (US)
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 15016736
- Report Number(s):
- PNNL-SA-42491; KP1102020; TRN: US200515%%1113
- Journal Information:
- Proteomics, Vol. 5, Issue 7; Other Information: PBD: 1 May 2005
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ABUNDANCE
CHO CELLS
DISEASES
EVALUATION
HAMSTERS
INSTABILITY
LIBERINS
MASS SPECTROSCOPY
MEMBRANE PROTEINS
MEMBRANES
PROTEINS
RADIATIONS
REGULATIONS
SPECTROSCOPY
PROTEIN SHEDDING
LOW-DOSE IRRADIATION
CROSS-SPECIES
MICROCARRIERS
FTICR
MASS SPECTROMETRY
SEQUEST
DISCRIMINANT SCORE