Abstract
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 standardized 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. For the discovery study, two sets of sera from patients with pancreatic cancer (n = 40) and healthy controls (n = 40) were obtained from two different clinical 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 evaluation was carried out by comparing two different bioinformatic strategies. Following proteome database search, the matching candidate peptide was verified by MALDI-TOF MS after specific antibody-based immunoaffinity chromatography and independently confirmed by an ELISA assay. 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 3884 to conventional clinical tumor markers (CA 19-9 and CEA) improved sensitivity and specificity, as shown by receiver operator characteristics curve analysis (AUROC(combined) = 1.00). Mass spectrometry-based m/z 3884 peak identification and following immunologic quantitation revealed platelet factor 4 as the corresponding peptide. MALDI-TOF MS-based serum peptidome profiling allowed the discovery and validation of platelet factor 4 as a new discriminating marker in pancreatic cancer.
Highlights
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
We identified and confirmed platelet factor 4 (PF4) as a potential marker peptide for pancreatic cancer using MALDI-TOF mass spectrometry (MS)–based clinical serum peptidome profiling with special consideration for the preanalytic preconditions and bioinformatic intricacies
The additional application of PF4 strongly improves www.aacrjournals.org the diagnostic power of conventional serum tumor marker panels consisting of CA 19-9 and carcinoembryonic antigen (CEA)
Summary
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. Conclusions: MALDI-TOF MS-based serum peptidome profiling allowed the discovery and validation of platelet factor 4 as a new discriminating marker in pancreatic cancer. No single clinical chemical marker meets the sensitivity and specificity criteria required for screening or stratification purposes [4] Established serum markers such as carbohydrate antigen CA 19-9 or carcinoembryonic antigen (CEA) are useful to monitor the course of disease on and off treatment, but they lack the prerequisites for screening and to estimate the prognosis of a patient [2, 5]. Based on matrix-assisted laser desorption/ionization time-of-flight peptidome profiling, the present study identifies platelet factor 4 as a discriminating serum marker in patients with pancreatic cancer. This might be of special relevance in the differential diagnosis of pancreatic cancer and pancreatitis
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