Untargeted LC-MS/MS profiling and semi-quantification of Gly-Pro-Yaa and Gly-Hyp-Yaa tripeptides in collagen hydrolysates

Gly-Pro-Yaa and Gly-Hyp-Yaa are characteristic triplet sequences in collagens, and tripeptides of both types are of interest for featuring proline hydroxylation sites and quality evaluation of commercial peptide products. Current LC-MS/MS multiple reaction monitoring (MRM) methods could only detect selected Gly-Xaa-Yaa tripeptides in collagen hydrolysates owing to the limit of MRM channels. Here, an approach for simultaneous screening and semi-quantifying Gly-Pro-Yaa and Gly-Hyp-Yaa tripeptides was developed based on LC-MS/MS precursor ion scan, which provides broader analyte coverage compared to MRM's target-restricted detection. The diagnostic fragment ions at m/z 127 and 143, as well as the precursor m/z ranges of 230–336 and 246−252, were effective for detecting Gly-Pro-Yaa and Gly-Hyp-Yaa tripeptides, respectively, and there were 8 Gly-Pro-Yaa tripeptides and 12 Gly-Hyp-Yaa tripeptides screened out from 25 commercial collagen tripeptide products. By calculating the relative response factors (0.59−1.7) of six candidates, Gly-Pro-Hyp and Gly-Hyp-Ala were chosen as the preferred calibration standards for semi-quantification of individual/total Gly-Pro-Yaa and Gly-Hyp-Yaa tripeptides, respectively, with sensitivity, linearity and reproducibility validated. Gly-Pro-Hyp, Gly-Pro-Ala, Gly-Pro-Arg, Gly-Hyp-Arg, Gly-Hyp-Ala, and Gly-Hyp-Lys were abundantly found with over 70 % of the commercial products containing 1 %–9 % of any one of them. Our study provides a novel LC-MS/MS method for the characterization of X-position proline hydroxylation in collagens and for the comprehensive profiling and semi-quantification of characteristic tripeptides in commercial collagen tripeptide products.