Interlysate comparison studies of human ovarian cancers: Development and application of a two-dimensional liquid separation/mass mapping technique

This work is focused on the interlysate comparison studies of human ovarian cancers. A novel two-dimensional liquid phase separation method, isoelectric focusing followed by nonporous reverse phase high performance liquid chromatography (IEF/NPS RP HPLC) has been developed as an alternative proteomic tool to the traditional 2-D gel electrophoresis analysis for separation of a large number of proteins from ovarian whole cell lysates. When HPLC is interfaced on-line to an electrospray ionization time-of-flight mass spectrometer (ESI TOF MS), highly accurate protein molecular weights (MWs) are obtained and 2-D pI/mass protein maps have been generated. The 2-D images can be used to monitor the protein expression changes in cancer cells versus normal cells. Fractions from HPLC can be collected, enzymatically digested and analyzed by matrix-assisted laser desorption-ionization (MALDI) TOF MS and tandem MALDI MS/MS peptide mass mapping. Proteins can then be identified using Ms-Fit and MASCOT database searching.; Three ovarian cell lines have been studied in this work, of which one is the normal ovarian surface epitheliam (OSE), and the other two are ovarian adenocarcinoma cell lines. By using the 2-D mass mapping method and various mass spectrometry techniques, accurate MWs together with the pI value and the corresponding hydrophobicity (HPLC retention time) are used to tag each protein so that protein expression can be compared in these interlysate studies, including comparisons between fractions from three cell lines. Furthermore, a complete MW map of ES2 ovarian carcinoma cells has been generated, which can serve as a standard map against which other ovarian carcinoma cell lines can be compared. With the aid of MS/MS analysis, the percentage of the proteins identified has been improved from around 35% to 55%. Using the three pieces of information, protein mass, pI and hydrophobicity, around 500 unique proteins have been identified with high confidence. Among the proteins identified, there are a few potential cancer markers, including tumor proteins and proteins whose overexpression is often associated with malignancy. The detection of cancer markers is essential to the early diagnosis of the disease and effective treatment for the purpose of improving the quality of human life.