Serum peptidome profiling revealed platelet factor 4 as a potential discriminating Peptide associated with pancreatic cancer.
OBJECTIVE
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.


METHODS
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.


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 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.


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.
Fiedler
Georg Martin
GM
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany.
Leichtle
Alexander Benedikt
AB
Kase
Julia
J
Baumann
Sven
S
Ceglarek
Uta
U
Felix
Klaus
K
Conrad
Tim
T
Witzigmann
Helmut
H
Weimann
Arved
A
Schütte
Christof
C
Hauss
Johann
J
Büchler
Markus
M
Thiery
Joachim
J
D014408:Biomarkers, Tumor / Q000097:blood; D001798:Blood Proteins / Q000032:analysis*; D018395:CA-19-9 Antigen / Q000097:blood; D002272:Carcinoembryonic Antigen / Q000097:blood; D003937:Diagnosis, Differential; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D010190:Pancreatic Neoplasms / Q000097:blood* / Q000175:diagnosis; D010978:Platelet Factor 4 / Q000097:blood*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D014408:Biomarkers, Tumor; D001798:Blood Proteins; D018395:CA-19-9 Antigen; D002272:Carcinoembryonic Antigen; D010978:Platelet Factor 4