Analysis of hydroxyproline isomers and hydroxylysine by reversed-phase HPLC and mass spectrometry

Collagens, the most abundant mammalian proteins, contain a high content of hydroxylated amino acids, such as, 3- and 4-cis-/trans-hydroxyproline (Hyp) and 5-hydroxylysine (Hyl). Whereas the global content of 4-Hyp was studied by amino acid analysis, no technique to determine all five hydroxyamino acids simultaneously in collagens has been reported. Here, we report the separation of all five hydroxyamino acids as well as two Hyp epimers from all other proteinogenic amino acids after derivatization with N2-(5-fluoro-2,4-dinitrophenyl)-l-valine amide (l-FDVA) by RPC-UV-ESI-MS. The general applicability of this method is shown for three Hyp-containing peptides as well as collagen type I.