Objective: To investigate the differential salivary protein expression profiles between children with severe early childhood caries (S-ECC) and caries-free (CF) children at the age of 3 years. Methods: We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with weak cation exchange magnetic beads, and peptide mass fingerprints were created by scanning mass spectrometry signals. Salivary samples from 20 children were analyzed (10 for each group). Results: Eleven protein peaks were significantly different (p < 0.05) between the two groups. Eight of these peaks were higher in the S-ECC group and three were higher in the CF group. To establish a diagnostic model for discrimination between the two groups, we chose three peptides (3,186.2, 3,195.8 and 3,324.8 Da) that exhibited the best fitted curve, by which the two groups were better separated when compared with other combinations. Conclusions: The salivary biomarkers identified revealed significant differences between the CF and the S-ECC group. Our results provide novel insight into the salivary protein profile of preschool-age children with dental caries and may lead to the development of a new strategy for screening high-risk populations.

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