Dental caries is a major worldwide oral disease afflicting a large proportion of children. As an important host factor of caries susceptibility, saliva plays a significant role in the occurrence and development of caries. The aim of the present study was to characterize the healthy and cariogenic salivary proteome and determine the changes in salivary protein expression of children with varying degrees of active caries, also to establish salivary proteome profiles with a potential therapeutic use against dental caries. In this study, unstimulated saliva samples were collected from 30 children (age 10–12 years) with no dental caries (NDC, n = 10), low dental caries (LDC, n = 10), and high dental caries (HDC, n = 10). Salivary proteins were extracted, reduced, alkylated, trypsin digested and labeled with isobaric tags for relative and absolute quantitation, and then they were analyzed with GO annotation, biological pathway analysis, hierarchical clustering analysis, and protein–protein interaction analysis. Targeted verifications were then performed using multiple reaction monitoring mass spectrometry. A total of 244 differentially expressed proteins annotated with GO annotation in biological processes, cellular component and molecular function were identified in comparisons among children with varying degrees of active caries. A number of caries-related proteins as well as pathways were identified in this study. As compared with caries-free children, the most significantly enriched pathways involved by the up-regulated proteins in LDC and HDC were the ubiquitin mediated proteolysis pathway and African trypanosomiasis pathway, respectively. Subsequently, we selected 53 target proteins with differential expression in different comparisons, including mucin 7, mucin 5B, histatin 1, cystatin S and cystatin SN, basic salivary proline rich protein 2, for further verification using MRM assays. Protein–protein interaction analysis of these proteins revealed complex protein interaction networks, indicating synergistic action of salivary proteins in caries resistance or cariogenicity. Overall, our results afford new insight into the salivary proteome of children with dental caries. These findings might have bright prospect in future in developing novel biomimetic peptides with preventive and therapeutic benefits for childhood caries.

Human saliva is a protein-rich, easily accessible source of potential biomarkers for the diagnosis of oral and systemic diseases. However, little is known about the changes in salivary proteome associated with aging of patients with dental caries. Here, we applied isobaric tags for relative and absolute quantitation (iTRAQ) in combination with multiple reaction monitoring mass spectrometry (MRM-MS) to characterize the salivary proteome profiles of subjects of different ages, presenting with and without caries, with the aim of identifying age-related biomarkers for dental caries. Unstimulated whole saliva samples were collected from 40 caries-free and caries-susceptible young adults and elderly individuals. Salivary proteins were extracted, reduced, alkylated, digested with trypsin and then analyzed using iTRAQ-coupled LC–MS/MS, followed by GO annotation, biological pathway analysis, hierarchical clustering analysis, and protein–protein interaction analysis. Candidate verification was then conducted using MRM-MS. Among 658 salivary proteins identified using tandem mass spectrometry, 435 proteins exhibited altered expression patterns in different age groups with and without caries. Of these proteins, 96 displayed age-specific changes among caries-susceptible adults and elderly individuals, and were mainly associated with salivary secretion pathway, while 110 age-specific proteins were identified among healthy individuals. It was found that the age factor caused significant variations and played an important role in both healthy and cariogenic salivary proteomes. Subsequently, a total of 136 target proteins with complex protein–protein interactions, including 14 age-specific proteins associated with caries, were further successfully validated using MRM analysis. Moreover, non-age-specific proteins (histatin-1 and BPI fold-containing family B member 1) were verified to be important candidate biomarkers for common dental caries. Our proteomic analysis performed using the discovery-through-verification pipeline revealed distinct variations caused by age factor in both healthy and cariogenic salivary proteomes, highlighting the significance of age in the great potential of saliva for caries diagnosis and biomarker discovery.