Serum Peptidome Profiling Revealed Platelet Factor 4 as a Potential Discriminating Peptide Associated With Pancreatic Cancer

Purpose: Mass spectrometry-based serum peptidome profiling is a promising tool to identify novel disease-associated biomarkers, but is limited by preanalytic factors and the intricacies of complex data processing. Therefore, we investigated whether standard- ized sample protocols and new bioinformatic tools combined with external data validation improve the validity of peptidome profiling for the discovery of pancreatic cancer- associated serum markers. Experimental Design: For the discovery study, two sets of sera from patients with pan- creatic cancer (n = 40) and healthy controls (n = 40) were obtained from two different clin- ical centers. For external data validation, we collected an independent set of samples from patients (n = 20) and healthy controls (n = 20). Magnetic beads with different surface functionalities were used for peptidome fractionation followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Data evalua- tion was carried out by comparing two different bioinformatic strategies. Following pro- teome database search, the matching candidate peptide was verified by MALDI-TOF MS after specific antibody-based immunoaffinity chromatography and independently con- firmed by an ELISA assay. Results: Two significant peaks (m/z 3884; 5959) achieved a sensitivity of 86.3% and a specificity of 97.6% for the discrimination of patients and healthy controls in the external validation set. Adding peak m/z 3884to conventional clinical tumor markers (CA 19-9 and CEA) improved sensitivity and specificity, as shown by receiver operator characteristics curve analysis (AUROCcombined = 1.00). Mass spectrometry-based m/z 3884peak identifi- cation and following immunologic quantitation revealed platelet factor 4as the corre- sponding peptide. Conclusions: MALDI-TOF MS-based serum peptidome profiling allowed the discovery and validation of platelet factor 4as a new discriminating marker in pancreatic cancer.