Abstract
Hydroxyproline (Hyp) is an imino acid posttranslationally 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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
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 J 3:1609–1617
Pihlajaniemi T, Myllyla R, Kivirikko KI (1991) Prolyl 4-hydroxylase and its role in collagen-synthesis. J Hepatol 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, West KA, Dodson WS, Hulmes JD (1997) Amino acid analysis in current protocols in protein science. In: Coligan JE, Dunn BM, Ploegh HL, Speicher DW, and Wingfield PT, (eds) Wiley New York, pp. 11.9.1–11.9.42
Bellon G et al (1984) Separation and evaluation 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
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Langrock, T., Hoffmann, R. (2019). Analysis of Hydroxyproline in Collagen Hydrolysates. In: Alterman, M. (eds) Amino Acid Analysis. Methods in Molecular Biology, vol 2030. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9639-1_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9639-1_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9638-4
Online ISBN: 978-1-4939-9639-1
eBook Packages: Springer Protocols