This proposal involves the development of new methodology for detection and sequencing of cancer-related target proteins at very low levels. In order to identify possible cancer-related proteins a novel highly accurate 2-dimensional mass map will be used for the differential analysis of the protein content of tumor cells. The method will use a total liquid phase 2-0 separation of proteins interfaced on-line to an electrospray (ESI) - time-of-flight (TOF) mass spectrometer (MS) detector. A batch liquid phase IEF-based method will be used as the first phase of separation while nonporous silica(NPS) reversed phase (RP) HPLC will be used as the second phase of separation. The use of the total liquid separation will be shown to have major advantages for on-line interfacing to mass spectrometry compared to 2-0 gel methods. It will be shown that between 1000-2000 unique protein masses over a mass range of 5-100 kDa and a pl between 3-10 can be detected. Of more significance is that for each unique protein peak, the molecular weight (MW) can be determined within less than a 100 ppm using current commercial ESI-TOFMS technology. The result is a highly accurate virtual 2-0 protein map in which pl is mapped against exact MW. The method will provide a quantitative protein map in digital form which can be used to search for changes in expression of proteins in cancer cells. It will be demonstrated that by using this method with a differential display program developed in our lab that subtle changes in protein expression can be observed in terms of quantitation and in terms of changes in MW. Also changes in phosphorylation patterns and other modifications or the presence of protein isoforms can be readily detected even up to 100 kDa using the ESI-TOFMS method. In addition, the eluent of the 2-D liquid separations can be collected as isolated proteins which can then be further analyzed to provide identification and detailed sequence analysis. Initial identification will be performed using MALDI-TOF MS peptide mapping and database searching. The use of the MW, pl, peptide map and species will be shown to provide excellent matches against the database. The target marker proteins identified will be sequenced using enzymatic digestion with on-line capillary electrophoresis interfaced via microelectrospray to the ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOFMS). Full on-line tandem MS can be achieved for every peak to provide detailed analysis of modified isoforms of the protein to search for changes in phosphorylation patterns as a function of cancer progression. The methodology will ultimately be applied to marker proteins identified and isolated by the 2-0 liquid separations/mass mapping method for breast and ovarian cancer samples. The method will provide a means of identifying potential markers that change as a function of cancer and also some of the most detailed information on aberrant signaling associated with cancer progression.
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