Summary of 3Hyp occupancy in the (GPP)n of type I and II collagen α-chains.

The table shows the average number of 3Hyp residues per (GPP)n motif with the percentage of α-chains containing at least one 3Hyp residue per (GPP)n given in parentheses. The percentage of each posttranslational variant was determined based on the ratio of the heights of the m/z peaks. For example, the human tendon α1(I) (GPP)n tryptic peptide, TGDAGPVGPPGPPGPPGPPGPPSAGFDFSFLPQPPQEK, was found to be a mix of eight distinct molecular species giving a hydroxylation (±16 Da) ladder, each representing a posttranslational variant (Figure 2). The molecular location of the each hydroxylated residue (3Hyp, 4Hyp and Hyl) was determined using MS/MS (Figure S1). The C-terminal lysine was predominantly hydroxylated in all Achilles tendons. In this scroll, the 1270.73+ m/z (peptide species containing four 3Hyp residues and five 4Hyp) represents 9% of the total population and the other variations are as follows: 1265.93+ (three 3Hyp residues and five 4Hyp, 10%); 1260.53+ (three 3Hyp residues and four 4Hyp, 13%); 1254.63+ (two 3Hyp residue and four 4Hyp, 19%); 1249.63+ (one 3Hyp residue and four 4Hyp, 16%); 1244.13+ (no 3Hyp residues and four 4Hyp, 18%); 1238.63+ (no 3Hyp residues and three 4Hyp residue, 10%); 1233.13+ (no 3Hyp residues and two 4Hyp residue, 5%). From these percentages, the average number of 3Hyp residues was estimated per α-chain. In this example the calculation is (4×9%)+(3×10%)+(3×13%)+(2×19%)+(1×16%) = mean content of 1.6 3Hyp per α1(I) from human tendon. The 3Hyp content in mouse tendon type I collagen was observed to vary markedly with animal age, in the range between one and two 3Hyp residues per (GPP)n as indicated in the table.