Salivary peptidome profiling for diagnosis of severe early childhood caries

Abstract

Background: Severe early childhood caries (s-ECC), which has quite high prevalence among children, is a widespread problem with significant impacts among both developing and developed countries. At present, it is widely known that no early detective techniques and diagnostic tests could have high sensitivity and specificity when using for clinical screening of s-ECC. In this study, we had applied magnetic bead (MB)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to screen distinctive candidate biomarkers of this disease, so as to establish protein profiles and diagnostic models of s-ECC.

Methods: Firstly, we used the technique mentioned above to detect specifically expressed peptides in saliva samples from ten children with s-ECC, separately at the time point of before, 1 and 4 weeks after dental treatment. Then a diagnostic model for s-ECC was established with the K nearest-neighbour method, which was validated in another six children in the next stage of study. After that, linear ion trap-orbitrap-mass spectrometry (LTQ-Orbitrap-MS) was performed to identify which of the proteins in saliva might be the origination of these peptides.

Results: We found that seven peptide peaks were significantly different when comparing the three time points, among them two were higher, while other five were lower in the pre-treatment s-ECC group compared with post-treatment. The sensitivity and specificity of the diagnostic model we built were both 83.3 %. Two of these peptides were identified to be segments of histatin-1, which was one important secretory protein in saliva.

Conclusions: Hereby we confirmed that MB-based MALDI-TOF MS is an effective method for screening distinctive peptides from the saliva of junior patients with s-ECC, and histatin-1 may probably be one important candidate biomarker of this common dental disease. These findings might have bright prospect in future in establishing new diagnostic methods for s-ECC.

Keywords: Biomarker; Early diagnosis; Histatin-1; MALDI-TOF MS; Proteomics; Saliva; s-ECC.

Fig. 1

The entire mass spectra of the peptide samples in the range 1000–10,000 Da. These data were from ten patients with s-ECC before and after treatment. Green, untreated; red, treated for 1 week; blue, treated for 4 weeks. m/z, mass-to-charge ratio

Fig. 2

Three-dimensional m/z ratio-intensity maps. Those showed the seven significantly different peptides at 1723.7, 1851.4, 1886.5, 2331, 2995.7, 1370.4, and 3310.0 Da. Green, untreated; red, treated for 1 week; blue, treated for 4 weeks

Fig. 3

Histograms and scatter plots from the three groups. a Histogram of the mass spectra from the three groups, showing increased expression of peptides at 1723.7, 1851.4, 1886.5, 2331.0, and 2995.7 Da, and decreased expression of those at 1370.4 and 3310.0 Da, in s-ECC patients treated for 1 and 4 weeks. (*P < 0.05; **P < 0.01). b Histogram of the mass spectra at 1723.7 Da of all patients from the three groups, showing increased expression in s-ECC patients treated for 1 and 4 weeks. (*P < 0.05; **P < 0.01). c Scatter plots of the three groups established by combining the peptides at 1723.7 and 2995.7 Da, and showing well-distinguished curve fitting

Fig. 4

Salivary histatin-1 levels increased in children with s-ECC treated for 4 weeks. These levels were compared with before treatment. a Western blotting reveals the presence of histatin-1 in each salivary sample. b The relative density of the histatin-1 bands, revealing that levels of salivary histatin-1 are higher in children with s-ECC after treated for 4 weeks compared with before treatment  (**P < 0.01)