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).

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Journal Journal of Proteome Research
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van Huizen, N.A, Burgers, P.C, Saintmont, F., Brocorens, P., Gerbaux, P, Stingl, C, … Luider, T.M. (2019). Identification of 4-Hydroxyproline at the Xaa Position in Collagen by Mass Spectrometry. Journal of Proteome Research, 18(5), 2045–2051. doi:10.1021/acs.jproteome.8b00930