Identification of 4‑Hydroxyproline at the Xaa Position in Collagen by Mass Spectrometry

Collagen
has a triple helix form, structured by a [-Gly-Xaa-Yaa-] repetition, where
Xaa and Yaa are
amino acids. This repeating unit can be post-translationally modified
by enzymes, where proline is often hydroxylated into hydroxyproline
(Hyp). Two Hyp isomers occur in collagen: 4-hydroxyproline (4Hyp, Gly-Xaa-Pro,
substrate for 4-prolyl hydroxylase) and 3-hydroxyproline
(3Hyp, Gly-Pro-4Hyp, substrate for 3-prolyl hydroxylase). If 4Hyp is
lacking at the Yaa position, then Pro at the Xaa position
should remain unmodified. Nevertheless, in literature 41 positions
have been described where Hyp occurs at the Xaa position (?xHyp) lacking
an adjacent 4Hyp. We report four additional positions in liver and
colorectal liver metastasis tissue (CRLM). We studied the sequence
commonalities between the 45 known positions of ?xHyp. Alanine and
glutamine were frequently present adjacent to ?xHyp. We showed that
proline, position 584 in COL1A2, had a lower rate of modification
in CRLM than in healthy liver. The isomeric identity of ?xHyp, that
is, 3- and/or 4Hyp, remains unknown. We present a proof of principle
identification of ?xHyp. This identification is based on liquid chromatography
retention time differences and mass spectrometry using ETD-HCD fragmentation,
complemented by ab initio calculations. Both techniques identify ?xHyp
at position 584 in COL1A2 as 4-hydroxyproline (4xHyp).