Discovery and Absolute Quantification of Water‐Soluble Signature Peptides via LC–MS/MS Based on Proteomics for Quality Evaluation of Goat Horn

INTRODUCTION: Goat horn is a primary substitute for the endangered Saiga antelope horn in traditional medicines. Current quality control (QC) methods lack specificity to effectively detect economically motivated adulteration with horns from other Bovidae species. OBJECTIVE: This study is aimed at discovering the species-specific peptides and to develop a reliable mass spectrometry-based method for authenticating goat horn and quantifying its signature peptides. METHODS: A label-free proteomics strategy was employed to characterize the tryptic digests across five Bovidae species: goat horn (originated from Capra hircus), sheep horn (Ovis aries), buffalo horn (Bubalus bubalis), cattle horn (Bos taurus), and yak horn (Bos grunniens) on a nanoLC-Orbitrap mass spectrometry and screen signature peptides. A novel ultrasound-assisted simultaneous extraction-enzymolysis (UASEE) method was developed for sample preparation. An absolute quantification for seven goat horn peptides was established on UHPLC-MS/MS. Twenty-two batches of goat horn samples were analyzed. RESULTS: Thirteen signature peptides enabling unambiguous differentiation of five Bovidae species were identified. The UASEE method reduced the sample preparation time from over 24-6 h. The quantification method demonstrated satisfactory linearity (r ≥ 0.997), precision (RSD < 8.2%), repeatability (RSD < 5.8%), stability (RSD < 9.2%), and accuracy (recoveries 84.6%-114.2%, RSD < 13.9%). Based on the quantification results, GHpep 7 (mean 0.224 mg/g) and GHpep 11 (mean 0.137 mg/g) were proposed as the optimal QC markers due to high abundance and minimal batch variability (RSD < 15%). CONCLUSION: This work provides a platform for signature peptide discovery and absolute quantification using UASEE and LC-MS/MS for quality evaluation of goat horn. It effectively addresses adulteration challenges and establishes a foundation for QC standards of goat horn.