Abstract
Hydroxyproline (Hyp) is an imino acid post-translationally formed by sequence-specific hydroxylases in the repeating collagen Gly–Xaa–Yaa triad present in all collagen types of all species. In both Xaa- and Yaa-positions, Pro is the most common residue, often oxidized to 4-Hyp in the Yaa- and rarely to 3-Hyp in the Xaa-positions. Here, we describe the qualitative and quantitative analysis of 3- and 4-Hyp-isomers by separating the free imino acids either with hydrophilic interaction chromatography (HILIC) or after derivatization with reversed-phase chromatography (RPC). In both cases, the compounds were detected by electrospray ionization mass spectrometry (ESI-MS).
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References
Ramachandran GN, Kartha G (1955) Structure of collagen. Nature 176:593–595
Rich A, Crick FHC (1955) Structure of Collagen. Nature 176:915–916
Crick FHC (1954) A Structure for Collagen. J Chem Phys 22:347–348
Kivirikko KI, Myllyla R, Pihlajaniemi T (1989) Protein Hydroxylation – Prolyl 4-Hydroxylase, An Enzyme with 4 Cosubstrates and A Multifunctional Subunit. FASEB Journal 3:1609–1617
Pihlajaniemi T, Myllyla R, Kivirikko KI (1991) Prolyl 4-Hydroxylase and Its Role in Collagen-Synthesis. J Hepatology 13:S2–S7
Myllyharju J (2003) Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis. Matrix Biol 22:15–24
Gryder RM, Lamon M, Adams E (1975) Sequence position of 3-hydroxyproline in basement membrane collagen. Isolation of glycyl-3-hydroxyprolyl-4-hydroxyproline from swine kidney. J Biol Chem 250:2470–2474
Clifton IJ et al (2001) Structure of proline 3-hydroxylase. Evolution of the family of 2-oxoglutarate dependent oxygenases. Eur J Biochem 268:6625–6636
Crabb JW et al (1997) Amino Acid Analysis in Current Protocols in Protein Science (John Wiley & Sons, Inc.)
Bellon G et al (1984) Separation and Evalua-tion of the Cis and Trans Isomers of Hydroxyprolines – Effect of Hydrolysis on the Epimerization. Anal Biochem 137:151–155
Dziewiatkowski DD, Hascall VC, Riolo RL (1972) Epimerization of Trans-4-Hydroxy-L-Proline to Cis-4-Hydroxy-D-Proline During Acid-Hydrolysis of Collagen. Anal Biochem 49:550–558
Langrock T, Czihal P, Hoffmann R (2006) Amino acid analysis by hydrophilic interaction chromatography coupled on-line to electrospray ionization mass spectrometry. Amino Acids 30:291–297
Alpert AJ (1990) Hydrophilic-Interaction Chromatography for the Separation of Peptides, Nucleic-Acids and Other Polar Compounds. J Chromatogr 499:177–196
Bruckner H, Keller-Hoehl C (1990) HPLC Separation of DL-amino acids derivatized with N2-(5-fluoro-2,4-dinitrophenyl)-L-amino acid amides. Chromatographia 30:621–629
Langrock T, García-Villar N, Hoffmann R (2007) Analysis of Hydroxyproline by Reversed-Phase HPLC and Mass Spectrometry. J Chromatogr B 847:282–288
Kindt E et al (2003) Determination of hydroxyproline in plasma and tissue using electrospray mass spectrometry. J Pharm Biomed Anal 33:1081–1092
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Langrock, T., Hoffmann, R. (2012). Analysis of Hydroxyproline in Collagen Hydrolysates. In: Alterman, M., Hunziker, P. (eds) Amino Acid Analysis. Methods in Molecular Biology, vol 828. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-445-2_21
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DOI: https://doi.org/10.1007/978-1-61779-445-2_21
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