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

Abstract

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-Nhydroxysuccinimidyl 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.

In-depth correlation analysis demonstrates that 4-hydroxyproline at the Yaa position of Gly-Xaa-Yaa repeats dominantly stabilizes collagen triple helix

“…2 A). The amount of the unusual 4Hyp was especially high in earthworm collagen, as reported previously [3] . When invertebrate collagens were removed from this dataset, Xaa position 4Hyp actually negatively correlated with T d ( r = −0.666, p = 2.56E−03).…”

“…We further investigated these correlations by analyzing Pro and Hyp via discrimination of their position in the Gly-Xaa-Yaa repeat sequences using our LC–MS methodology [3] . While large proportions of Pro and 4Hyp were present at the Xaa and Yaa positions, respectively, small amounts of 4Hyp/3Hyp at the Xaa position and Pro at the Yaa position were also detected in all animal species ( Supplementary Table 3 ), consistent with our understanding of collagen prolyl hydroxylation 1 , 2 , 3 . Total content of imino acids at respective positions largely varied among animal species (70.2–112.8 residues/1000 residues at the Xaa position and 67.8–110.5 residues/1000 residues at the Yaa position).…”

“…Although it was expected that Gly-4Hyp-Yaa tripeptides would negatively correlate with T d based on the results of 4Hyp assigned to the Xaa position, no correlation was detected for Gly-4Hyp-Ala or Gly-4Hyp-Gly. Since the method used to quantitate the position-specific contents of Pro/Hyp judges the location of the imino acids from the positional relationship to Gly, 4Hyp at the Yaa position of Gly-Gly-Yaa-Gly sequences was counted as occupying both the Xaa and Yaa positions [3] . The amount of Gly-Gly-4Hyp was relatively high compared to the two Gly-4Hyp-Yaa tripeptides analyzed here, suggesting that the irregular sequence with Yaa position 4Hyp significantly affected the estimated values of Xaa position 4Hyp.…”

“…The primary structural feature of collagen is repeating Gly-Xaa-Yaa amino acid sequences where Pro and hydroxyproline (Hyp) are frequently found at the Xaa and Yaa positions, respectively. In general, most post-translational hydroxylation reaction of Pro residues occurs at the Yaa position generating 4Hyp, while 4Hyp and 3Hyp are rarely generated at the Xaa position in Gly-Xaa-Yaa and Gly-Xaa-4Hyp sequences, respectively 1 , 2 , 3 . Three polypeptide chains of collagen form a triple helical structure, which is important not only for the structural integrity but also for the biological functions [4] .…”

“…Mass spectrometric approaches with protease digestion have been increasingly used for comprehensive identification of prolyl hydroxylation sites in collagen 16 , 17 , 18 , 19 , 20 , 21 , but it is difficult to totally evaluate the degree of the modification with discrimination of the sequence position. Recently, we developed a novel method to quantitatively analyze positional distribution of Pro and Hyp by LC–MS with partial acid hydrolysis [3] . The method enables determination of the imino acid contents as residues/1000 amino acid residues with discrimination of the position (Xaa or Yaa) and hydroxylation type (Pro, 4Hyp, or 3Hyp).…”