Recently paleoproteomics, the study of ancient proteins recovered from archaeological and paleontological samples, has been radically developed. Paleoproteomic studies usually use type 1 collagen (COL1), the predominant protein in bones and teeth (dentine) of vertebrates. In this study, we evaluate the protein recovery rate from these two different tissues to foster the applications of this method to a broader range of paleontological specimens. We sampled Pleistocene elephantid specimens recovered in the Bisan Seto area, Japan: one rib, one ulna, and three incisors. To evaluate protein preservation in the samples, we used total protein and collagen colorimetry, electrophoresis (SDS-PAGE), and MALDI-TOF mass spectrometry for amino acid sequence determination of the sampled protein. SDS-PAGE on the rib and ulna showed two distinct bands under 37 kilodaltons, which were not observed on incisor samples and the negative control. The determined amino acid sequences of the bone samples based on mass spectrometry were identical to that of COL1 peptides of Mammut americanum, the only elephantid COL1 sequence in the protein sequence database that we used. The colorimetric analysis quantified the total protein and collagen extracted from the bones (128 – 168 and 12.7 – 14.1 mg/g) and the incisors (0.00 – 1.32 and 0.00 – 0.79). The results of SDS-PAGE and mass spectrometry indicate that we are successfully recovering endogenous fossil proteins from bone samples. The result of the colorimetric analysis demonstrates that the protein recovery rate is significantly better in the bone samples than in the dentine samples. This pattern can be explained by the difference in microscopic structures between bones and dentine; numerous and consistently arranged dentine tubules in teeth facilitate the loss of proteins during diagenesis. Studies of protein extraction from fossil elephantid samples often use incisors, but this study suggests that the use of bone instead of teeth would improve the extraction result of fossil proteins. This will greatly expand the breadth of samples available for future analyses, including, for example, bonebed material represented by fragmentary bones and edentulous taxa.
