Raw mass spectrometry data for "Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes"

<strong>Abstract:</strong> Lysyl hydroxylase 2 (LH2) is a member of LH family of enzymes (LH1-3) that catalyze the hydroxylation<br> of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in Bruck syndrome,<br> fibrosis and cancer metastasis. Previously, we proposed LH2 as a telopeptidyl LH for type I collagen and<br> this specific function is now generally accepted. However, several fundamental questions remain<br> unanswered: 1, Is LH2 responsible for both N- (α1 and 2 chains) and C-telopeptidyl (α1 chain) Lys<br> hydroxylation? 2, Is LH2 involved in the helical Lys hydroxylation? 3, what are the functional<br> consequences when LH2 is completely lacking? To answer these questions, we generated LH2-null MC3T3<br> cells (LH2 KO) using CRISPR/Cas9, and extensively characterized the molecular and fibrillar phenotypes<br> of type I collagen. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hyl^ald)-derived crosslinks<br> were completely absent from LH2 KO collagen with concomitant increases in the Lys^ald-derived crosslinks.<br> Mass spectrometric analysis revealed that, in LH2 KO type I collagen, telopeptidyl Lys hydroxylation<br> was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a sitespecific<br> manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl.<br> Furthermore, prolyl 3-hydroxylation was slightly increased in LH2 KO type I collagen. In LH2 KO samples,<br> collagen solubility was markedly increased, fibril diameters are significantly diminished, and<br> mineralization severely impaired. Together, these data underscore the critical role of LH2-catalyzed<br> collagen modifications and consequent cross-linking in matrix stability, organization and mineralization.