Title
Proteome analysis of high affinity mouse BALB/cA saliva-1 proteins to hydroxyapatite 

Keywords
Mouse saliva, Hydroxyapatite, Dental caries

Description
In order to identify a protein candidate for dental caries prevention, a proteomic analysis of hydroxyapatite (HAP) adsorbed mouse saliva proteins has been performed

Sample: Whole saliva was collected from two strains of 8-week-old male conventional BALB/cA mice. The mice were stimulated with 0.5 ml of 0.5 mg/ml pyrocalopinsulfate and 0.5 ml of 2 mg/ml isoproterenol to collect saliva for five minutes. Whole saliva samples were fractionated using a Hydroxyapatite-HPLC column (HAP-HPLC, 3 mm x 10 cm). Elution of HAP-HPLC adsorbed proteins was carried out with a potassium phosphate buffer solution (pH 6.6) at a flow rate of 1 ml/min, stepwise with holding for 20 minutes at 50, 70, 100, 150, 200 and 400 mM or with a linear gradient from 50 to 400 mM. Each eluted fraction was desalted using an ultrafiltration membrane (Amicon Ultra - 15, Merck Millipore). The eluted HAP-HPLC fractions of BALB/cA were labeled with Amersham CyDye DIGE Fluor Cy3 minimal dye (GE Healthcare, Tokyo, Japan), and two-dimensional gel electrophoresis performed. The first dimension was IEF with a gradient from pI 3 to pI 10 using IPGphor IEF System (Amersham Biosciences, Amersham, UK), the second dimension was SDS-PAGE. The proteins were visualized by fluorescence detection with Cy 3 at 580 nm, using Typhoon Imager. The spots excised from the 2D-PAGE were subjected to S-pyridine-ethylation treatment, digested with trypsin overnight, and then analyzed by MALDI-TOF MS using AXIMA-QIT (Shimadzu, Kyoto, Japan). To identify the proteins MASCOT analysis was performed with the obtained MS spectral data.