Comparison of three methods for enamel protein extraction in different developmental phases of rat lower incisors

Abstract

Protein extraction methods [urea, trichloroacetic acid (TCA), and acetic acid] were compared for protein recovery from rat incisor developing enamel in the S phase (intermediate/late secretion), M1 phase (early maturation), M2 phase (intermediate maturation), and M3 phase (final maturation). We compared the protein recoveries with the percentage of enamel matrix dry weight burnt off by incineration. Our results indicate that TCA and urea were equally efficient for the extraction of S-stage proteins (85% and 90% recovery, respectively), while urea was the best for M1-stage proteins (92% recovery), and TCA the best for M2-stage (99% recovery) and M3-stage (60% recovery) proteins. The other methods yielded less than 30% recovery in comparison to incineration for M2 and M3 stages. The fact that urea extraction works well in the S and M1 stages and not thereafter is probably related to the changes in the proteins during enamel development and the amount of mineral that needs to be dissolved. TCA is the single method that effectively recovered proteins from all developmental stages of the rat incisor enamel.

Extraction and sequencing of human and Neanderthal mature enamel proteins using MALDI-TOF/TOF MS

“…That enamel proteins can be successfully extracted from modern mature enamel has already been confirmed by earlier studies (Porto et al, 2006), but these studies also confirm that the recoverable amount of protein from enamel in the later maturation stages is very low. The MS and MS/MS data obtained from the medieval and clinical samples in this study suggest that extraction and sequencing of proteins from mature tooth enamel using MALDI-TOF/TOF MS is possible.…”

“…Following drilling and enamel-dentine separation, a modified version of the method described in Porto et al (2006) was used on all samples to extract the enamel proteins. z1-2 mg of enamel powder was placed in a 0.5 ml eppendorf and 10% trichloroacetic acid (TCA) (1 mg enamel powder 200 ml À1 TCA) containing a set of protease inhibitors (Complete Mini; Roche Applied Sciences) was added.…”

New techniques for the recovery of small amounts of mature enamel proteins

“…The enamel powder was dissolved in 12% trichloroacetic acid (TCA) in a proportion of 200 mL acid/mg enamel powder. One hour later, sodium deoxycolate was added in a final concentration of 200 mg/mL, as described earlier (Porto et al, 2006). After enamel dissolution at 0 C, the solution was centrifuged at 2500 Â g at 4 C for 45 min.…”

“…After enamel dissolution at 0 C, the solution was centrifuged at 2500 Â g at 4 C for 45 min. The pellet was ressuspended in 200 mL of a 6 M urea solution containing a wide range of protease inhibitors, as described before (Porto et al, 2006).…”

“…In the latter case (Nielsen-Marsh et al, 2009), protein extraction from the tooth enamel was accomplished from w2 mg of enamel powder from each of the Neanderthalis teeth as the starting material, and the precipitation protocol was based on the use of TCA (Nielsen-Marsh et al, 2009), as proposed for the recovery of proteins from mature rodent enamel (Porto et al, 2006). An older study also used acid dissolution to extract and characterize mature bovine enamel proteins.…”