ABSTRACT
The 80% mortality rate of pancreatic-cancer (PC) makes early diagnosis a challenge. Oral fluids (OF) may be considered the ultimate body fluid for non-invasive examinations. We have developed techniques to improve visualization of minor OF proteins thereby overcoming major barriers to using OF as a diagnostic fluid. The aim of this study was to establish a short discriminative panel of OF biomarkers for the detection of PC. Unstimulated OF were collected from PC patients and controls (n = 30). High-abundance-proteins were depleted and the remaining proteins were analyzed by two-dimensional-gel-electrophoresis and quantitative dimethylation-liquid-chromatography-tandem mass-spectrometry. Label-free quantitative-mass-spectrometry analysis (qMS) was performed on 20 individual samples (n = 20). More than 100 biomarker candidates were identified in OF samples, and 21 had a highly differential expression profile. qMS analysis yielded a ROC-plot AUC value of 0.91 with 90.0% sensitivity and specificity for a combination of five biomarker candidates. We found a combination of five biomarkers for PC. Most of these proteins are known to be related to PC or other gastric cancers, but have never been detected in OF. This study demonstrates the importance of novel OF depletion methodologies for increased protein visibility and highlights the clinical applicability of OF as a diagnostic fluid.
Subject(s)
Biomarkers, Tumor/metabolism , Pancreatic Neoplasms/diagnosis , Pancreatic Neoplasms/metabolism , Proteomics , Saliva/metabolism , Aged , Case-Control Studies , Electrophoresis, Gel, Two-Dimensional , Humans , Methylation , Middle Aged , Neoplasm Proteins/metabolismABSTRACT
OBJECTIVES: The aim of this study was to examine whether triple depletion of salivary-α-amylase (sAA), albumin (Alb) and immunoglobulins G (IgGs) may improve the visualization capability of proteins in two-dimensional gel electrophoresis (2-DE) of oral fluids (OF). SUBJECTS AND METHODS: Oral fluids from healthy volunteers were subjected sequentially to sAA removing device followed by application to an Alb and IgG immunoaffinity column (triple depletion). The depleted OF samples were analyzed using SDS-PAGE followed by 2-DE and protein identification using ion-trap mass spectrometry (MS). RESULTS: This specific triple depletion technique unmasked spots never visualized before. A total of 36 new spots were observed after depletion (348 vs 312 before depletion). Moreover, 58 spots showed more than twofold increase intensity after depletion. In the 60-69kDa area, the depletion procedure unmasked 14 proteins including HSP70, LTA4H, L-Plastin, Desmoplakin that are known to be involved in disease pathogenesis. CONCLUSION: The ability to selectively remove and elute the most abundant OF proteins visualized on the 2-DE represents an important step in the characterization of human OF. The better visualization and gel resolution achieved will improve quantification abilities in 2-DE and in tag-MS leading to better identification of disease-specific biomarkers. We further analyzed the eluted Alb and IgGs isoforms suggesting a new methodology venue for OF.
