Mass Spectrometric Analysis of Cytoplasmic Ribosomal Proteins in Drug Resistant and Drug Susceptible Human Cell Lines

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Hays, Faith A
Fenselau, Catherine
This study examines changes in cytoplasmic ribosomes that accompany drug resistance in MCF-7 breast cancer cells. Differences in ribosomal protein composition between drug susceptible and drug resistant cell lines were examined. Ribosomes were isolated from mitoxantrone susceptible and mitoxantrone resistant MCF-7 cells. The acid extracted ribosomal proteins were subjected to optimized 2DGE using a "zoom" strip (pI 7-11) for the first dimension separation. Further optimization of 2DGE included the use of a 15mM DTT wick at the cathode end of the focusing tray, decreasing the protein loading amount and using large format gels for the second dimension. Forty-nine ribosomal proteins were identified in the drug susceptible cell line. Two novel protein isoforms of the proteins RPS3 and one novel isoform of RPS10 were identified in the drug resistant cell line. Methods for the extraction and detection of ribosomal proteins from the 2D gel were developed. The method of Mirza was modified and used to extract ribosomal proteins from the gel. The detection of these proteins was optimized by the use of 50% ACN/1.0% TFA to solubilize the MALDI matrix. In addition, the extracted protein solution was mixed 1:1 with 5% Triton X-100. Intact molecular weights were determined for 41 ribosomal proteins using high performance MALDI-TOF mass spectrometry. The average number of ribosomes per cell was determined for the drug susceptible, as well as the drug resistant cell line, and found to be unchanged.