Protein patterns expressed by tumor cells give clinically useful information about the metastatic and invasive potential of the tumor. Objectives: Our long-term objective is to measure protein expression patterns in biopsies and other small tissue samples using SELDI-TOF (surface enhanced laser desorption & ionization - time of flight) mass spectrometry (MS) to separate proteins by molecular weight and measure the amount of each protein. Our aim was to simplify the analysis of the tumor cell proteome by examining subgroups of proteins that bind to hydrophobic, anion-exchange, or cation-exchange surfaces on Ciphergen ProteinChip Arrays. Methods: Three tumor clones that differ in metastatic potential were extracted with high or low ionic strength media (Guanidine thiocyanate-HEPES-Triton X100 or Urea-Thiorea-CHAPS). Proteins were reduced and alkylated, concentrated by acid precipitation, washed, and dissolved in low or high ionic strength media. Samples were diluted, proteins were bound to the arrays, and unbound proteins and salts were removed by washing in buffer and water. Samples of three extracts from each clone were analyzed in duplicate on a Ciphergen PBS IIc spectrometer with an energy-absorbing sinapinic acid matrix. Results: A different protein spectrum was obtained for each clone on each array. A total of 52, 114, and 81 protein peaks in the 2,200 to 85,000 M/z range were detected in average spectra from H50, Q10, and CM52 arrays. Potential biomarkers included 36 polypeptides with peak intensities in the high potential clone that differed by more than 4-fold from those in the non-metastatic clone. M/z values of potential biomarkers that were positively correlated with metastatic potential were 12,100 (H50); 6,091, 12,104, 29945, 30,131 (Q10); 9,528 (CM10). Conclusions: The complex mixture of proteins in tumor cells extracts can be quickly separated into subgroups of hydrophobic, anionic, and cationic proteins for SELDI-TOF MS detection of possible tumor biomarkers. Supported by USPHS DE07258

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