Quantitative Analysis of the Positional Distribution of Hydroxyproline in Collagenous Gly-Xaa-Yaa Sequences by LC–MS with Partial Acid Hydrolysis and Precolumn Derivatization

Collagen is extensively modified by various enzymes, including prolyl hydroxylases. Pro residues at the Yaa position of repeating Gly-Xaa-Yaa amino acid sequences are mostly hydroxylated to 4-hydroxyproline (4Hyp), which is essential for the thermal stability of collagen triple helix. In contrast, Pro residues at the Xaa position are rarely modified to 3Hyp and 4Hyp, the biological function of which is poorly understood. Overall estimation of prolyl hydroxylation with discrimination of the position (Xaa or Yaa) and hydroxylation type (4Hyp or 3Hyp) has been difficult to perform using traditional methods. In the present study, we developed a novel position-specific analytical method featuring LC–MS detection of collagenous Gly-containing dipeptides, including Gly-Pro, Pro-Gly, Gly-4Hyp, Gly-3Hyp, and 4Hyp-Gly, after partial acid hydrolysis and precolumn derivatization using 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS). We performed acid hydrolysis at 55 °C with HCl/trifluoroacetic acid/water (2:1:1, v/v) to avoid peptide inversion and imbalanced peptide generation observed for collagenous model peptides. The positional distribution of Pro, 4Hyp, and 3Hyp can be calculated from the relative concentrations of the APDS-derivatized dipeptides, and in combination with amino acid analysis, we can determine their absolute contents at the Xaa and Yaa positions. Bovine type I, III, and V collagens were analyzed by the established method, and the amount of 4Hyp was higher than that of 3Hyp at the Xaa position in type I and III collagens. In addition, we clearly showed that collagen extracted from earthworm cuticles has an extremely high content of Xaa position 4Hyp, reaching over 10% of the total amino acids.