Abstract
BackgroundHuman 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.MethodsUnstimulated 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.ResultsAmong 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.ConclusionsOur 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.
Highlights
Human saliva is a protein-rich, accessible source of potential biomarkers for the diagnosis of oral and systemic diseases
We aimed to (1) identify the salivary proteomic profiles of individuals of different ages presenting with and without dental caries; (2) characterize the changes in salivary proteome influenced by age and caries susceptibility; and (3) seek for age-specific and non-age-specific proteins associated with dental caries
In an attempt to put our results to good use, we propose that these age-specific proteins determined from an analysis of proteome variations in whole saliva could provide suitability for caries biomarker screening among different age groups
Summary
Human saliva is a protein-rich, accessible source of potential biomarkers for the diagnosis of oral and systemic diseases. 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. Alterations in salivary protein composition can be monitored using diagnostics techniques and compared with other clinical parameters. Age-related variations in the salivary composition and gland morphology have been reported in healthy individuals [6]. A previous study compared the differences in the composition of the salivary proteome in healthy subjects stratified according to age by means of 2D-SDS-PAGE [8]. Another study reported no significant age-related changes in the salivary composition [9]. A thorough understanding of whole saliva is a prerequisite for its diagnostic utility
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