## 2 Results and Discussion
### Definition of Marker Peptide of Gelatins from Sika Deer  
Subsequently, gelatins commonly used in the pharmaceutical, food, and cosmetic industries, including donkey hide, bovine, porcine, and fish gelatin, were enzymatically digested, and high-resolution mass spectrometry data of the resulting peptide mixtures were obtained, following the same protocol as for sika deer gelatin. To verify the specificity of marker peptides for sika deer gelatin in the broader context of commonly used animal-derived gelatins, a comparative analysis was performed between the high-resolution mass spectrometry data and sequence information from public databases of the other animal species and the analytical data obtained for sika deer gelatin. To evaluate the presence of selected marker peptide candidates in all gelatin samples, the relative abundance of each candidate was calculated based on the peak area, using the trypsin-derived peptide peak (LGEHNIDVLEGNEQFINAAK, \(m/z\) 737.71, triple-charged), consistently detected across all samples, as a reference (Table 2). Due to significant differences in detection intensity between peptides P1-P12 and R1-R16, principal component analysis (PCA) was performed separately for the peptide P group and R group. As shown in the biplot (Figure 1), among the peptide P group, P1, P4, P5, P7, P9, P11, and P12 contributed to the identification of sika deer gelatin. Similarly, among the peptide R group, R1-R6, R9, R10, and R13-R16 were found to contribute to the identification of sika deer gelatin. Furthermore, to ensure the clear identification of sika deer gelatin, marker peptides were defined based on the following criteria. For P1-P12, peptides must show high detection intensity in all sika deer gelatin samples (peak area ratio relative to the trypsin-derived peptide > 3.0) and not be detected in at least two other animal-derived gelatin samples (peak area ratio < 1.0). For R1-R16, peptides must be detected in all sika deer gelatin samples (peak area ratio > 0.5) and not detected in at least two other animal-derived gelatin samples (peak area ratio < 0.1).  
Although commercial gelatin samples from horses, sheep, and goats (closely related ungulates to deer) were unavailable, the amino acid sequences of type I collagen \(\alpha\)1 and \(\alpha\)2 chains from these closely related ungulates were obtained from the UniProt database. Basic Local Alignment Search Tool (BLAST+, v2.16.0) searches were conducted to verify the uniqueness of the selected marker peptide candidates (Table 2). To enhance specificity, peptides with identical sequences across all three ungulates were excluded as marker peptides. As a result, among the 28 marker peptide candidates, P11, R2, R3, and R4 were defined as both specific to sika deer gelatin and exhibiting relatively high detection intensities. The combination of multiple marker peptides has been shown to improve the reliability and accuracy of species identification [25; 26]. In light of this, the selected set of  
Figure 1: Principal component analysis (PCA) of sika deer gelatin and commercially available gelatins with peptides (**A**) P1–P12 and (**B**) R1–R16.  
four marker peptides is considered well suited for distinguishing and verifying the use of gelatin from sika deer.