Quantitative Proteomic Analysis of the Effect of Fluoride on the Acquired Enamel Pellicle

Abstract

The acquired enamel pellicle (AEP) is a thin film formed by the selective adsorption of salivary proteins onto the enamel surface of teeth. The AEP forms a critical interface between the mineral phase of teeth (hydroxyapatite) and the oral microbial biofilm. This biofilm is the key feature responsible for the development of dental caries. Fluoride on enamel surface is well known to reduce caries by reducing the solubility of enamel to acid. Information on the effects of fluoride on AEP formation is limited. This study aimed to investigate the effects of fluoride treatment on hydroxyapatite on the subsequent formation of AEP. In addition, this study pioneered the use of label-free quantitative proteomics to better understand the composition of AEP proteins. Hydroxyapatite discs were randomly divided in 4 groups (n = 10 per group). Each disc was exposed to distilled water (control) or sodium fluoride solution (1, 2 or 5%) for 2 hours. Discs were then washed and immersed in human saliva for an additional 2 hours. AEP from each disc was collected and subjected to liquid chromatography electrospray ionization mass spectrometry for protein identification, characterization and quantification. A total of 45 proteins were present in all four groups, 12 proteins were exclusively present in the control group and another 19 proteins were only present in the discs treated with 5% sodium fluoride. Relative proteomic quantification was carried out for the 45 proteins observed in all four groups. Notably, the concentration of important salivary proteins, such as statherin and histatin 1, decrease with increasing levels of fluoride. It suggests that these proteins are repulsed when hydroxyapatite surface is coated with fluoride. Our data demonstrated that treatment of hydroxyapatite with fluoride (at high concentration) qualitatively and quantitatively modulates AEP formation, effects which in turn will likely impact the formation of oral biofilms.

Acidulated Phosphate Fluoride Application Changes the Protein Composition of Human Acquired Enamel Pellicle

“…However, the enamel surface modifications showed an effect in the quantity of some proteins, such as histatin-1, a recognized pellicle precursor that was found to be present in low abundance in enamel pellicle from slabs pretreated with APF. In a recent study, the same protein showed lower affinity for HA slabs pretreated with neutral fluoride solution than for HA with no treatment [Siqueira et al, 2012a].…”

“…onstrated that HA discs pretreated for 2 h with different concentrations of neutral fluoride solutions modulate the pellicle composition [Siqueira et al, 2012a]. Although all the results are valuable, from a clinical perspective, the effect of APF on pellicle composition is still not known.…”

Changes in the Proteomic Profile of Acquired Enamel Pellicles as a Function of Their Time of Formation and Hydrochloric Acid Exposure

“…We employed an in vivo design that had been used before to evaluate proteins in the AEP that are resistant to removal by acids that mimic extrinsic erosion (citric acid) and caries (lactic acid) [Delecrode et al, 2015b]. Thus, the results obtained here are more realistic than those described in studies involving in vitro [Siqueira et al, 2012a;Masson et al, 2013;Custodio et al, 2015] or in situ [Delecrode et al, 2015a;Delius et al, 2017] protocols. Since our study was conducted in vivo, it was not possible to use other methods of collection, such as sonication or treating the samples with strong detergents that are cytotoxic, such as Triton X-100 or SDS, as has been done in in situ studies [Delius et al, 2017].…”

Triethylene Glycol Up-Regulates Virulence-Associated Genes and Proteins in Streptococcus mutans

“…Supernatant were carefully removed, separated in aliquots of 50 μL and stored at -80°C. The total protein concentration in each sample was assessed by the micro bicinchoninic acid (Micro BCA) assay [49]. Equal amounts of protein (20 μg) from both experimental and control groups were dried by a rotary evaporator, denatured and reduced for 2 hours by the addition of 200 μL of 4 M urea, 10 mM dithiothreitol (DTT), and 50 mM NH 4 HCO 3 , pH 7.8.…”

“…Peptide separation and mass spectrometric analyses were carried out as described previously [49]. The MS/MS spectra were analysed against the streptococcal protein database (Swiss Prot and TrEMBL, Swiss Institute of Bioinformatics, Geneva, Switzerland, http://ca.expasy.org/sprot/) using SEQUEST algorithm in Proteome Discoverer 1.3 software (Thermo Scientific, San Jose, CA, USA) [49].…”