Assaying proline hydroxylation in recombinant collagen variants by liquid chromatography-mass spectrometry.
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.
Department of Chemical Engineering and Materials Science, University of California, Irvine, CA 92697, USA.
S W Polly
SW
Chan
John
J
Greaves
Nancy A
NA
Da Silva
Szu-Wen
SW
Wang
D002853:Chromatography, Liquid / Q000379:methods*; D003094:Collagen / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D013058:Mass Spectrometry / Q000379:methods*; D011392:Proline / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D003094:Collagen; D011392:Proline