Monitoring the Glycosylation Status of Proteins Using Raman Spectroscopy
Monitoring the Glycosylation Status of Proteins Using Raman Spectroscopy
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DOI：
10.1021/ac2012009
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发表时间：
2011-08-01
影响因子：
7.4
通讯作者：
Goodacre, Royston
中科院分区：
文献类型：
--
作者：
Brewster, Victoria L.;Ashton, Lorna;Goodacre, Royston
基于蛋白质的生物药物正越来越广泛地用作治疗剂，并且这些生物药物的表征构成了显著的分析挑战。特别是，监测翻译后修饰（PTM），如糖基化，是这种表征的一个重要方面，因为这些聚糖可以强烈影响这些生物活性药物的稳定性，免疫原性和药理学。拉曼光谱是一种强大的工具，在生物加工竞技场中具有许多新兴的应用。虽然该技术在蛋白质科学中有着相对丰富的历史，但直到最近才研究了拉曼光谱用于评估翻译后修饰，包括磷酸化，乙酰化，三甲基化和泛素化。在这项调查中，我们开发的第一次拉曼光谱结合多元数据分析，包括主成分分析和偏最小二乘回归，用于确定蛋白质的糖基化状态和定量的天然核糖核酸酶（RNase）A蛋白和RNase B糖蛋白的相对浓度的混合物。
Protein-based biopharmaceuticals are becoming increasingly widely used as therapeutic agents, and the characterization of these biopharmaceuticals poses a significant analytical challenge. In particular, monitoring posttranslational modifications (PTMs), such as glycosylation, is an important aspect of this characterization because these glycans can strongly affect the stability, immunogenicity, and pharmacolcinetics of these biotherapeutic drugs. Raman spectroscopy is a powerful tool, with many emerging applications in the bioprocessing arena. Although the technique has a relatively rich history in protein science, only recently has Raman spectroscopy been investigated for assessing posttranslational modifications, including phosphorylation, acetylation, trimethylation, and ubiquitination. In this investigation, we develop for the first time Raman spectroscopy combined with multivariate data analyses, including principal components analysis and partial least-squares regression, for the determination of the glycosylation status of proteins and quantifying the relative concentrations of the native ribonuclease (RNase) A protein and RNase B glycoprotein within mixtures.