Proteomic analysis for the identification of proteins related to methotrexate resistance

Methotrexate(MTX) is one of the most important and frequently used drugs in cancer therapy, but the efficacy of this drug is often compromised by the development of resistance in cancer cells. To seek and identify differentially expressed proteins related to MTX resistance and provide clues for the mechanism of MTX resistance, proteins from cell line MTX300 (resistant to 300 micromol/L MTX) and its control cell line 3T3R500 were separated by two-dimensional electrophoresis (2-DE). The colloidal Coomassie brilliant blue-stained 2-DE gels were subjected to image analysis, which revealed several spots with high levels of differential expression between MTX300 and 3T3R500. The protein spot with highest differential expression was submitted for tryptic peptide mass fingerprinting(PMF) for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MS analysis and database searches revealed it to be dihydrofolate reductase (DHFR), which was subsequently confirmed by Western blot. The result suggested that DHFR might play an important role in the MTX resistance.