Supplementary Material for: Proteomic Mapping of Dental Enamel Matrix from Inbred Mouse Strains: Unraveling Potential New Players in Enamel

<p>Enamel formation is a complex 2-step process by which proteins are secreted to form an extracellular matrix, followed by massive protein degradation and subsequent mineralization. Excessive systemic exposure to fluoride can disrupt this process and lead to a condition known as dental fluorosis. The genetic background influences the responses of mineralized tissues to fluoride, such as dental fluorosis, observed in A/J and 129P3/J mice. The aim of the present study was to map the protein profile of enamel matrix from A/J and 129P3/J strains. Enamel matrix samples were obtained from A/J and 129P3/J mice and analyzed by 2-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry. A total of 120 proteins were identified, and 7 of them were classified as putative uncharacterized proteins and analyzed in silico for structural and functional characterization. An interesting finding was the possibility of the uncharacterized sequence Q8BIS2 being an enzyme involved in the degradation of matrix proteins. Thus, the results provide a comprehensive view of the structure and function for putative uncharacterized proteins found in the enamel matrix that could help to elucidate the mechanisms involved in enamel biomineralization and genetic susceptibility to dental fluorosis.</p

Supplementary Material for: Proteomics of Secretory-Stage and Maturation-Stage Enamel of Genetically Distinct Mice

<p>The mechanisms by which excessive ingestion of fluoride (F) during amelogenesis leads to dental fluorosis (DF) are still not precisely known. Inbred strains of mice vary in their susceptibility to develop DF, and therefore permit the investigation of underlying molecular events influencing DF severity. We employed a proteomic approach to characterize and evaluate changes in protein expression from secretory-stage and maturation-stage enamel in 2 strains of mice with different susceptibilities to DF (A/J, i.e. ‘susceptible' and 129P3/J, i.e. ‘resistant'). Weanling male and female susceptible and resistant mice fed a low-F diet were divided into 2 F-water treatment groups. They received water containing 0 (control) or 50 mg F/l for 6 weeks. Plasma and incisor enamel was analyzed for F content. For proteomic analysis, the enamel proteins extracted for each group were separated by 2-dimensional electrophoresis and subsequently characterized by liquid-chromatography electrospray-ionization quadrupole time-of-flight mass spectrometry. F data were analyzed by 2-way ANOVA and Bonferroni's test (p < 0.05). Resistant mice had significantly higher plasma and enamel F concentrations when compared with susceptible mice in the F-treated groups. The proteomic results for mice treated with 0 mg F/l revealed that during the secretory stage, resistant mice had a higher abundance of proteins than their susceptible counterparts, but this was reversed during the maturation stage. Treatment with F greatly increased the number of protein spots detected in both stages. Many proteins not previously described in enamel (e.g. type 1 collagen) as well as some uncharacterized proteins were identified. Our findings reveal new insights regarding amelogenesis and how genetic background and F affect this process.</p