The purpose of this study was to identify biomarkers of pancreatic cancer in human serum. Stable isotope labeling of amino acids in cell culture (SILAC) was used to prepare a labeled proteome used as an internal standard in protein quantification by two dimensional liquid chromatography tandem mass spectrometry (2D-LC/MS/MS). A major obstacle to identifying meaningful biomarkers in pancreatic cancer is the presence of numerous acute-phase proteins. While the levels of many acute-phase proteins are increased in pancreatic cancer, they represent a systemic response, are not directly produced by pancreatic cancers, and lack the sensitivity and specificity of clinically useful biomarkers. The use of a stable isotope labeled proteome derived from a pancreatic cancer cell line as the internal standard makes it possible to focus only on proteins derived from pancreatic cancers, and to accurately quantitate their relative levels in human serum. CAPAN-2 cells were grown to near confluence in DMEM containing [13C6,15N1]leucine and [13C6,15N2]lysine (Cambridge Isotopes) and 10% dialyzed FBS. The cells were passaged at least five times. Conditioned media was collected every 2 days, pooled and aliquoted. These cells were subsequently grown in serum-free media supplemented with insulin, transferrin and selenium. Protein (1 mg) in the conditioned media from the final passage of the cells was trypsin digested and resulting peptides separated by preparative strong cation exchange LC into 40 fractions. Peptides in each fraction were analyzed by reversed-phase LC/MS/MS using a Finnigan LTQ linear ion trap mass spectrometer (Thermo Electron, San Jose, CA). Raw spectra were searched against the RefSeq Human database. Protein identifications were determined with the assistance of Peptide Prophet and Protein Prophet. A total of 376 unique proteins were identified. Analysis of the mass spectra revealed that incorporation of [13C6,15N1]leucine and [13C6,15N2]lysine into the proteome was > 99.5 %. Equal amounts of the labeled proteome were added as an internal standard to each of the individual human serum samples from patients with pancreatic cancer and those with benign pancreatic disease. Using stable isotope dilution 2D-LC/MS/MS a number of proteins identified in the conditioned media were increased in sera from patients with pancreatic cancer. A larger set of serum samples will be analyzed to determine the clinical utility of these potential biomarkers. Supported by NIH grant P30 CA16520 and NIH/NIDDK grant P32-DK-007066-29.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]