## 4 Conclusions  
The present study identified and validated characteristic marker peptide combinations that reliably distinguish sika deer gelatin from other animal-derived gelatins using LC-MS/MS analysis. By analyzing high-resolution mass spectrometry data from trypsin-digested peptide mixtures of gelatin samples and sequence information from public databases, the peptides P11 (GETGPAPAGPIGPVGAR), R2 (SGETGAS-GPP(+15.99)GFAGEK), R3 (TGQP(+15.99)GAVGPAGIR), and R4 (GPP(+15.99)GESGAAGP-AGPIGSR) were selected as species-specific marker peptides with high detection intensities in gelatin from sika deer antlers, hides, and bones. Notably, the marker peptide P11 has not been reported in other species of deer [32, 33, 34, 35, 36, 37]. Comparative analyses with commercially available gelatins confirmed the exclusivity of these marker peptides to sika deer, with no significant detection in gelatins derived from donkey hide, bovine, porcine, or fish sources. Among the selected markers, the peptide P11 exhibited consistently high intensity across all sika deer gelatin samples, making it a particularly robust indicator. In addition, the peptides R2, R3, and R4 demonstrated distinctive patterns linked to the anatomical origin of the sika deer gelatin, suggesting their potential for further refinement in differentiating among sika deer gelatin types. In contrast, gelatins from other species presented distinct peptide profiles, underscoring the specificity of the marker peptide set for sika deer. The established marker peptides provide a reliable and efficient tool for verifying the authenticity of sika deer gelatin, thereby supporting its safe and effective use as a health-promoting resource.  
**Supplementary Materials:** The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/molecules30071528/s1](https://www.mdpi.com/article/10.3390/molecules30071528/s1)]([https://www.mdpi.com/article/10.3390/molecules30071528/s1](https://www.mdpi.com/article/10.3390/molecules30071528/s1)), Figure S1: Total ion chromatograms (TIC) in positive ion mode of sika deer gelatin and commercially available gelatins; Figures S2-S4: Overlay of total ion chromatograms (TIC) in positive ion mode for triplicate digestion experiments of sika deer gelatin; Figures S5-S7: Extracted ion chromatograms (XIC) of marker peptides in three different lots of sika deer gelatin; Figures S8-S11: Mass and product ion spectrum for marker peptides P11, R2, R3, and R4; Table S1: Identified peptides in sika deer antler gelatin; Table S2: Identified peptides in sika deer bone gelatin; Table S3: Identified peptides in sika deer skin gelatin.  
**Author Contributions:** K.O.: Methodology, investigation, formal analysis, writing--original draft, project administration. A.N.: Investigation. M.Z.: Formal analysis, writing--review and editing. D.L.: Methodology, writing--review and editing. T.K.: Methodology, writing--review and editing. W.L.: Conceptualization, methodology, supervision, project administration, writing--review and editing. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This research received no funding.  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** All new research data were presented in this contribution.  
**Acknowledgments:** The authors are grateful to [PERSON] (Hokkaido Rokubiken Co., Ltd.) for providing the research materials. All authors express their appreciation to the editorial board and reviewer for their suggestions and efforts in reviewing this paper. The authors also appreciate the editor for his assistance during the review process.  
**Conflicts of Interest:** The authors declare no competing financial interests.