Abstract
BACKGROUND: Cholangiocarcinoma (CCA) is a highly aggressive malignancy frequently diagnosed at advanced stages due to the lack of effective biomarkers for early-stage detection. This study aimed to identify serum peptide biomarkers capable of distinguishing healthy individuals, benign bile duct disease, different stages of CCA, and hepatocellular carcinoma (HCC). METHODS: We analyzed 306 serum samples, comprising 50 healthy controls, 53 benign bile duct disease, 138 CCA, and 65 HCC patients. Peptide mass fingerprints (PMFs) were generated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and a random forest model was applied to classify these groups based on PMF patterns. To complement this analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on pooled serum samples to identify representative peptides associated with each group. RESULTS: The 94 PMFs from MALDI-TOF MS yielded high classification performance within the study cohort, with an internal accuracy of 97.96. LC-MS/MS analysis of pooled samples identified representative peptides associated with each disease group, providing complementary information to PMF-based classification. Importantly, peptide biomarkers with high classification performance were associated with specific patient groups. CONCLUSION: These findings suggest that serum peptide biomarkers may serve as potential signatures to support CCA diagnosis, staging, and risk stratification, offering a non-invasive approach that warrants further investigation.