Quantitative Peptidomic profiling of caprine and bovine Milk after simulated gastrointestinal digestion: A multidimensional analytical strategy integrating UHPLC-Q-TOF-MS and QqQ-MS

Caprine and bovine milk have been widely used as a supplementary nutritional source during early infancy. This study established an integrated peptidomics workflow combining in vitro digestion with multidimensional mass spectrometry (UHPLC-Q-TOF-MS/QqQ-MS) to decode species-specific peptide release patterns. Systemic peptidomic profiling identified 1728 and 1088 unique peptides in caprine and bovine skimmed milk digests, respectively, with β-casein-derived peptides dominating both systems (CHMP:126 vs BTMP:116). Two β-casein regions (P90-K112 and M159-L180) were identified as proteolytic hotspots generating structurally diverse peptides. Through relative-to-absolute quantification cascades, ten signature sequences were validated: LYQEPVLGPVR, SDIPNPIGSENSGK, YQEPVLGPVR, INNQFLPYPY, YPVEPFTESQSL, QGPIVLNPW, SLAMAASDISLL, LTEEEK, DMPIQAF, VLVLDTDYK, with species-dependent abundance: YQEPVLGPVR peaked in BTMP (0.84 mg·g⁻¹) versus DMPIQAF in CHMP (1.39 mg·g⁻¹). These sequence-resolved quantitative maps reveal the species-specific peptide clusters and provided templates for designing hypoallergenic infant formulas or cardiovascular nutraceuticals. This study provided data support for the functional research of dairy products in vivo.