Proteomic studies of human breast cancer cell lines using multidimensional separation techniques and time-of-flight mass spectrometry

In this work, a two-dimensional liquid-phase separation technique coupled with time-of-flight mass spectrometry (TOF-MS) is presented for proteomic studies of human breast cancer. Preparative liquid isoelectric focusing was performed as the first dimension, and nonporous silica reverse-phase high-performance liquid chromatography (NPS RP-HPLC) as the second dimension. NPS RP-HPLC was directly coupled with electrospray ionization (ESI) TOF-MS to obtain accurate intact protein molecular weights. Furthermore, mass fingerprinting and tandem mass spectrometry were performed on tryptic digests of proteins using matrix-assisted laser desorption ionization (MALDI) TOF-MS.; Human breast epithelial cell lines were studied from a xenograft model of progressive proliferative breast disease. This model consists of the normal MCF10A breast epithelial cells and includes both premalignant and fully malignant variants derived from MCF10A. Proteomic analysis was performed for contribution to breast epithelial protein databases, identification of breast cancer biomarkers, and structural characterization of oncoproteins. This research has resulted in positive identification of over fifty proteins in normal and fully malignant cells. Some of these proteins were found to be differentially expressed between the two cell lines, and represent potential breast cancer biomarkers. Furthermore, studies were conducted on the effect of estradiol on protein expression in premalignant cells. Proteins were identified, including some known oncoproteins, that were differentially expressed in the premalignant cells with estradiol (MCF10AT1E), compared to the MCF10AT and the fully malignant CA1d.cl1 cells. Structural analysis was performed on the oncoproteins p60 c-src and posttranslational modification differences were found between MCF10AT1E and MCF10AT1.