The prostate secretes a range of proteins related to its function, including proteolytic enzymes, acid phosphatase (ACPP), and prostate-specific antigen (PSA). In prostate tumors, some of these proteins are known to increase or decrease in the amount secreted. To test the application of secretory vesicle isolation to frozen human surgical specimens, samples were obtained from patients with prostate cancer, with institutional review body (IRB) approval and patient consent. Tumor and normal tissue were separated as described  and the content of the secretory apparatus was isolated. Proteins were digested with trypsin and analyzed by LC-MS in order to compare expression between each normal and tumor pair from all six patients. Significantly differentially expressed peptides were identified. Four of the best known prostate cancer-associated secreted proteins were found to be up-regulated, as expected, in the tumors. PSA, ACPP, kallekrin 2 (KLK2), and macrophage migration inhibitory factor (MIF) are all low-abundance proteins in the blood, at levels ranging from 0.2 to 3.5ng/mL in normal individuals. Detection of these proteins by direct LC-MS analysis of plasma would not be possible, yet was readily accomplished by examination of the secretory pathway. Two of these proteins, PSA and MIF, were also evaluated by commercial ELISA in the plasma of the same patients. The levels observed in the secretory vesicles by unlabeled mass spectrometry were directly comparable to levels in the plasma from the same patients, with correlation coefficients of 0.69 and 0.74, respectively . Thus, analysis of differentially expressed proteins in the secretory apparatus is predictive of relative expression in the plasma. This correlation means that discovery can be done in the highly concentrated milieu of the vesicles, while verification and validation studies can be conducted with more sensitive antibody-based assays directly in blood.
In addition to the well-known prostate cancer markers, other known cancer-related proteins were found, for a total of 40 proteins with known cancer association. A further 20 proteins were identified as differentially expressed in the tumors that were not previously known to have a cancer association. The known function of the proteins typically fit well with their observed expression patterns. For example, of 11 proteins involved with sugar metabolism, all were found to go up in the tumors. On the other hand, of nine proteins involved with contraction and adhesion, six were found to be expressed at lower levels in the tumor samples than in the matching normal tissue.
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