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首页> 外文期刊>BMC Biotechnology >Assaying proline hydroxylation in recombinant collagen variants by liquid chromatography-mass spectrometry
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Assaying proline hydroxylation in recombinant collagen variants by liquid chromatography-mass spectrometry

机译：液相色谱-质谱法测定重组胶原蛋白变体中脯氨酸的羟化反应

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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.
机译：背景技术重组胶原蛋白及其规定的变体的制备在组织再生，细胞-基质相互作用研究以及细胞外基质的基础生化和生物物理研究中具有巨大潜力。重组表达需要脯氨酸羟基化，这是翻译后修饰，对于赋予稳定性和结构至关重要。但是，这些修饰不是典型的细菌或酵母表达系统固有的。此外，就选择性和灵敏度而言，检测低水平的4-羟基脯氨酸具有挑战性。结果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脯氨酸的羟基化。在不同的酿酒酵母表达系统中测试了该测定法，以评估脯氨酰-4-羟化酶和胶原之间的基因比例对羟化程度的影响。这些系统使用了由寡核苷酸从头合成的人类胶原蛋白III基因。 LC-MS分析不需要衍生化，仅使用皮摩尔样品，并且可以测量重组胶原蛋白和天然胶原蛋白中脯氨酸的羟化水平，范围约为0％至40％。通过LC-MS获得的羟化值与使用常规氨基酸分析方法获得的羟化值一样准确。结论建立了一种简便，无衍生化的LC-MS方法，该方法可准确确定不同酵母表达系统中脯氨酸羟基化的百分比。使用该测定法，我们确定胶原蛋白与羟化酶基因拷贝比例较高的系统产生的羟化百分比较低，这表明需要特定平衡的基因比例才能获得更高的羟化水平。

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