? Prostate cancer is the most common non-cutaneous cancer in men in the United States and the second leading cause of cancer related death in men. While a serum marker, PSA, has been used for cancer screening, disagreement exists regarding the appropriate use of this test due to problems with sensitivity and specificity. Identification of other new proteins released by prostate cancer cells may allow more informed treatment decisions. We propose here a rapid, specific, and inexpensive strategy to identify and validate protein markers for prostate cancer screening. Our strategy achieves specificity by focusing exclusively on plasma membrane proteins because they are frequently released from cells into the bloodstream. We utilize a highly specific chemistry to capture membrane glycoproteins from four diverse culture models of human prostate cancer to generate an ensemble of target glycopeptides. To enable quantification, we generate isotopically heavy versions of these peptides by concatenating the DMA sequences for the tryptic glycopeptides in an artificial gene for the expression of this peptide string as a protein in bacteria, a strategy called QCAT. Bacteria will be grown in 15NH4CI, which produces complete incorporation and therefore a """"""""heavy"""""""" version of the artificial protein. The QCAT protein will be purified, quantified, and enzymatically digested into heavy peptides with the enzyme trypsin. Well will spike our heavy proteins into mouse serum as part of a dilution series and use multiple reaction monitoring (MRM), a high sensitivity mass spectrometry based method, to identify and quantify these peptides in serum from mouse models of prostate cancer. If successful this method will be applied to human serum from patients with prostate cancer using the same heavy QCAT peptides. Peptides identified in cases of prostate cancer but not in control samples indicate that the peptide is a predictor of prostate cancer. Antibody based tests, useful in clinical testing, will be created for the proteins that contain these predictive peptides and further testing of human serum will be performed. These tests could have beneficial public health effects by allowing the early detection of cancer as well as predict disease behavior. ? ? ?
