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

doi:10.1186/1472-6750-12-51

BMC Biotechnol. 2012; 12: 51.

Published online 2012 August 17. doi: 10.1186/1472-6750-12-51

PMCID: PMC3443662

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

S W Polly Chan,¹ John Greaves,² Nancy A Da Silva,¹ and Szu-Wen Wang¹

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

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

Corresponding author.

S W Polly Chan: swchan/at/uci.edu; John Greaves: jgreaves/at/uci.edu; Nancy A Da Silva: ndasilva/at/uci.edu; Szu-Wen Wang: wangsw/at/uci.edu

Received April 17, 2012; Accepted July 13, 2012.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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

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