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Open Access Highly Accessed Methodology article

Assaying proline hydroxylation in recombinant collagen variants by liquid chromatography-mass spectrometry

S W Polly Chan1, John Greaves2, Nancy A Da Silva1* and Szu-Wen Wang1*

Author Affiliations

1 Department of Chemical Engineering and Materials Science, University of California, Irvine, CA, 92697, USA

2 Department of Chemistry, University of California, Irvine, CA, 92697, USA

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BMC Biotechnology 2012, 12:51  doi:10.1186/1472-6750-12-51

Published: 17 August 2012

Abstract

Background

The fabrication of recombinant collagen and its prescribed variants has enormous potential in tissue regeneration, cell-matrix interaction investigations, and fundamental biochemical and biophysical studies of the extracellular matrix. Recombinant expression requires proline hydroxylation, a post-translational modification which is critical for imparting stability and structure. However, these modifications are not native to typical bacterial or yeast expression systems. Furthermore, detection of low levels of 4-hydroxyproline is challenging with respect to selectivity and sensitivity.

Results

We have developed a new liquid chromatography-mass spectrometry (LC-MS) method to evaluate proline hydroxylation in recombinant collagen. This assay was tested in different Saccharomyces cerevisiae expression systems to evaluate the effect of gene ratio between prolyl-4-hydroxylase and collagen on the extent of hydroxylation. These systems used a human collagen III gene that was synthesized de novo from oligonucleotides. The LC-MS assay does not require derivatization, uses only picomoles of sample, and can measure proline hydroxylation levels in recombinant and native collagen ranging from approximately 0% to 40%. The hydroxylation values obtained by LC-MS are as accurate and as precise as those obtained with the conventional method of amino acid analysis.

Conclusions

A facile, derivatization-free LC-MS method was developed that accurately determines the percentage of proline hydroxylation in different yeast expression systems. Using this assay, we determined that systems with a higher collagen-to-hydroxylase gene copy ratio yielded a lower percentage of hydroxylation, suggesting that a specifically balanced gene ratio is required to obtain higher hydroxylation levels.

Keywords:
Hydroxyproline; Liquid chromatography-mass spectrometry; LC-MS assay; Recombinant collagen