The proteome profile of the human osteosarcoma U2OS cell line. The human osteosarcoma U2OS cell line is one of the first generated cell lines and is used in various areas of biomedical research. Knowledge of its protein expression is limited and no comprehensive study on the proteome of this cell line has been reported to date. Proteomics technology was used in order to analyse the proteins of the U2OS cell line. Total protein extracts were separated by two-dimensional gel electrophoresis (2-DE) and analysed by matrix-assisted laser desorption ionisation-mass spectrometry (MALDI-MS) and MALDI--MS-MS following in-gel digestion with trypsin and, finally, protein identification was carried out by peptide mass fingerprint (PMF) and post source decay (PSD), respectively. Approximately 3,000 spots were excised from two 2-DE gels and were analysed, resulting in the identification of 237 different gene products. The majority of the identified proteins were enzymes, regulatory proteins and RNA-associated proteins, while leukocyte markers and oncogenes were also present. Our findings include 11 protooncogenes (FKBP4, SRC8, PSD10, FUBP1, PARK7, NPM, PDIA1, OXRP, SET, TCTP and GRP75) related to the cancerous state of the U2OS cell line. The U2OS 2-DE database provides the basis for future protein studies.