A strategy based on mass spectrometry proteomics is used to discover biomarkers for prostate and bladder cancer. The markers targeted are proteins secreted by the cancer cells and/or cancer-associated cells. Tumor tissue specimens are obtained from surgically resected organs after pathology. The specimens are digested by collagenase into single cells in a serum-free media. The digestion media, termed COL, is made cell-free by centrifugation, and it contains proteins made by the cells. A matched non-cancer specimen is processed similarly for comparative analysis. Differentially expressed protein species in the cancer and non-cancer COL pairs are screened by a method termed glycopeptide capture followed by tandem mass spectrometry for identification. Since virtually all secreted proteins are post-translationally modified by the addition of carbohydrate groups this method selectively analyzes these proteins. Quantification is achieved by isotopic labeling of the peptides, i.e. light isotopes for cancer and heavy isotopes for non-cancer. Candidate markers will be validated by Western blotting of the COL samples and immunohistochemistry if specific antibodies are available, or by in situ nucleic acid probe hybridization. This marker discovery process is illustrated by TIMP1, found in our initial experiments. The feasibility of proteomic analysis of urine samples to detect markers will be done. Preliminary experiments have generated a human urinary proteome of 239 protein species. Urine samples will be obtained from cancer patients and age-matched volunteers. The goal is to develop an assay for cancer associated protein markers that can be detected in voided urine for the diagnosis of prostate and bladder cancer. ? ? ?

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project--Cooperative Agreements (U01)
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Special Emphasis Panel (ZCA1-SRRB-E (O1))
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Kagan, Jacob
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University of Washington
Schools of Medicine
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