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

Abstract

BackgroundThe 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.ResultsWe 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.ConclusionsA 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.

Nanoscale architecture and cellular adhesion of biomimetic collagen substrates

“…The collagen gene was inserted in the CEN/ARS plasmid (YCpMCOL-trp) as previously described. 12 To hydroxylate prolines in the collagen protein, two human hydroxylase subunits, human prolyl-4-hydroxylase α-subunit cDNA (α, BC035813) and human PDI cDNA (β, BC029617), were integrated consecutively into the yeast genome using two-piece PCR integration to generate strains BYα1β1 and BYα2β2 as previously described. 13 BYα1β1 and BYα2β2 contain one copy and two copies of each of the hydroxylase subunits, respectively.…”

Collagen analysis with mass spectrometry

“…For LC–MS analysis, hydrolysis with hydrochloric acid is required (Yang et al, ; Wang et al, ; Metcalfe et al, ; Chan et al, ); GC–MS analysis requires an additional derivatization step to measure 4Hyp (Tredget et al, ). For LC–MS analysis, glycylphenylalanine (Chan et al, ) and N ‐methyl‐ l ‐proline (Yang et al, ; Wang et al, ; Metcalfe et al, ) may serve as internal standards. For MS analysis, however, it is recommended to use a stable isotope‐labeled version of the analyte of interest (Stokvis, Rosing, & Beijnen, ; Lanckmans et al, ).…”

LC–MS/MS Identification of the O-Glycosylation and Hydroxylation of Amino Acid Residues of Collagen α-1 (II) chain from Bovine Cartilage

“…46 The identification of glycopeptides from type I and II collagen using hydrazide-based chemistry enrichment in conjunction with LC-MS/MS was recently described. 47 Also, the levels of proline hydroxylation in recombinant collagen variants was recently investigated by LC-MS. 48 Yang et al 49 have comprehensively characterized the hydroxylation and glycosylation of type V collagen by LC-MS/MS, employing CID, ETD, higher-energy collision dissociation (HCD), and high order tandem MS. They identified a large number of HyP residues in Gly-Xaa-Yaa at both Xaa and Yaa positions.…”