Department of Chemical Engineering and Materials Science, University of California, Irvine, CA 92697, USA.
BMC Biotechnol. 2012 Aug 17;12:51. doi: 10.1186/1472-6750-12-51.
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.
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.
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.
重组胶原蛋白及其规定变体的制造在组织再生、细胞-基质相互作用研究以及细胞外基质的基本生化和生物物理研究方面具有巨大的潜力。重组表达需要脯氨酸羟化，这是一种翻译后修饰，对于赋予稳定性和结构至关重要。然而，这些修饰不是典型的细菌或酵母表达系统的天然产物。此外，4-羟脯氨酸的低水平检测在选择性和灵敏度方面具有挑战性。
我们开发了一种新的液相色谱-质谱（LC-MS）方法来评估重组胶原蛋白中的脯氨酸羟化。该测定法在不同的酿酒酵母表达系统中进行了测试，以评估脯氨酰-4-羟化酶和胶原蛋白之间的基因比例对羟化程度的影响。这些系统使用从头合成的寡核苷酸合成的人胶原蛋白 III 基因。LC-MS 测定法不需要衍生化，仅使用皮摩尔级的样品，并且可以测量重组和天然胶原蛋白中羟脯氨酸的羟化水平，范围约为 0%至 40%。通过 LC-MS 获得的羟化值与氨基酸分析的常规方法一样准确和精确。
开发了一种简单、无需衍生化的 LC-MS 方法，可准确测定不同酵母表达系统中脯氨酸羟化的百分比。使用该测定法，我们确定具有更高胶原蛋白-羟化酶基因拷贝数比例的系统产生的羟化百分比较低，这表明需要特定的平衡基因比例才能获得更高的羟化水平。
