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Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant neoplasms worldwide. Patients are often diagnosed at advanced stages with poor prognosis due to the absence of obvious early symptoms. Here, we applied a high-throughput serum peptidome analysis to identify circulating peptide markers of ESCC. Weak cationic exchange magnetic beads coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for two-stage proteotypic peptide profiling in complex serum samples collected from 477 cancer patients and healthy controls. We established a genetic algorithm model containing three significantly differentially expressed peptides at 1,925.5, 2,950.6 and 5,900.0 Da with a sensitivity and specificity of 97.00% and 95.92% in the training set and 97.03% and 100.00% in the validation set, respectively. The model's diagnostic capability was significantly better than SCC-Ag and Cyfra 21-1, especially for early stage ESCC, with an achieved sensitivity of 96.94%. Subsequently, these peptides were identified as fragments of AHSG, TSP1 and FGA by linear ion trap-orbitrap hybrid tandem mass spectrometry. Notably, increased tissue and serum levels of TSP1 in ESCC were verified and correlated with disease progression. In addition, tissue TSP1 was an independent poor prognostic factor in ESCC. In conclusion, the newly established circulating peptide panel and identified proteins could serve as potential biomarkers for the early detection and diagnosis of ESCC. Nevertheless, a larger cohort will be required for further unequivocal validation of their clinical application.

Novel circulating peptide biomarkers for esophageal squamous cell carcinoma revealed by a magnetic bead-based MALDI-TOFMS assay