Dentinogenesis: A proteomic approach to a dynamic process

Objectives: This study has applied proteomic technologies to further our understanding of the dynamic processes of dentinogenesis. The primary objective was to generate 2D protein maps to enable determination of the global synthesis, modification and turnover events which occur during the transition from an unmineralised predentine (PD) to mineralised dentine. Methods: ECM components from bovine incisors were dissociatively extracted in 4M GuCl, from the morphologically distinct phases, namely the PD, predentine-dentine (PD-D) interface and mineralised dentine. Soluble protein extracts (5mg) were examined for molecular weight components on 1D SDS-PAGE (12%). Protein profiling was performed by TCA precipitation of 100mg of protein, isoelectric focussing in the first dimension on immobiline pI 3-10 7cm strips and subsequent SDS-PAGE (12%) in the second dimension. Resultant 2D gels were stained by silver and hot Coomassie blue. Bands and protein spots of interest were excised, trypsin digested and analysed using MALDI-TOF mass spectrometry. Results: SDS-PAGE of extracts revealed common bands and demonstrated that the dentine extract contained less low molecular weight bands (<29kDa) than the PD and PD-D interface extracts. A complex array of proteins was apparent using 2D gel electrophoresis, with differential expression being noted between all three phases. The PD-D interface extract displayed less protein spots than the PD extract, which exhibited a greater proportion of acidic (pI<6) high molecular weight material (>100kDa). Preliminary MS-fingerprinting has identified several protein spots. Conclusions: This preliminary study has highlighted the wealth of information that can be obtained from a proteomic approach to understanding dentine mineralisation. Results have indicated various profiling changes, which may be degradation, post-translational modifications or synthetic events occurring during the transition from an unmineralised matrix to a mineralised dentine. Such changes may be key events and ultimately will enhance our knowledge of the mechanisms controlling mineral deposition.