Measurement of lysyl hydroxylase in fibroblasts by tandem mass spectrometry

Lysyl hydroxylase is an ascorbate dependent enzyme responsible for the posttranslation hydroxylation of lysyl residues on procollagen molecules. Several isoforms of Lysyl hydroxylase, encoded by different genes, have been identified. Lysyl hydroxylase 1, encoded by PLOD1 gene, is impaired in Ehlers-Danlos Syndrome (EDS) Kyphoscoliotic type, known as EDS type VI. Impaired activity of lysyl hydroxylase leads to a decreased number of hydroxylysyl residue in collagen and to an abnormal formation of the most stable, hydroxylysine derived, pyridinium cross-links. There are four hydroxylation sites involved in cross-linking in collagen, one in each telopeptide region and two in the triple helicol region of the molecule. Little in known about hydroxylation at these specific sites in patients with EDS VI. Traditional methods to measure enzyme activity in fibroblast are neither sensitive nor specific enough to differentiate sites of collagen hydroxylation by specific lysyl hydroxylase isoforms. In this study, two synthetic 12 amino acids oligopeptides representing the telopeptide and the triple helix cross-linking site were used to substrate for the hydroxylation reaction in fibroblast. The two synthetic oligopeptides were characterized by the hydroxylation of the telopeptide oligopeptide was studied using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This novel method allowed us to evaluate the hydrosylation of the telopeptide oligopeptide in EDS VI and normal fibroblasts.