Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis electrospray time-of-flight mass spectrometry
Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis electrospray time-of-flight mass spectrometry
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DOI：
10.1002/elps.200600075
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发表时间：
2006-07-01
期刊：
影响因子：
2.9
通讯作者：
Neusuess, Christian
中科院分区：
文献类型：
--
作者：
Balaguer, Elvira;Demelbauer, Uwe;Neusuess, Christian
重组人促红细胞生成素 (rHuEPO) 的糖基化是一种翻译后过程，可改变血液中糖蛋白的生物活性、溶解度和寿命，并且在很大程度上取决于细胞类型和细胞培养条件。为了改进现有的药物开发或产品质量控制方法，需要一种快速、简单的方法来提供有关 rHuEPO 和其他糖蛋白中存在的聚糖的广泛碳水化合物信息。在这里，使用新型毛细管涂层开发了一种通过 CZE-ESI-TOF MS 表征完整 rHuEPO 糖型的改进方法，并与之前的研究进行了比较。两种方法都可以快速分离并结合单一蛋白质亚型的准确质量表征。新型动态涂层提供接近于零的 EOF 分离，从而实现更好的分离。这导致了质谱分辨率的提高和次要亚型的检测。为了将明确的碳水化合物组成分配给每个完整的糖型，开发了一种用于酶促释放的未衍生 N-聚糖的 CZE-ESI-MS 分离方法。 TOF MS 具有较高的质量准确度和分辨率，可以正确识别聚糖。因此，乙酰化、氧化、硫酸化、甚至 NH2 交换 OH 等聚糖修饰均已成功表征。蛋白质骨架分子量的信息已与肽分析（揭示有关 O-糖基化的信息）和聚糖分析的结果相结合，包括检测尚未描述的含有四个触角和五个唾液酸的聚糖。这允许将整体糖基化组成明确分配给完整 rHuEPO 糖型获得的分子质量。
Glycosylation of recombinant human erythropoietin (rHuEPO) is a post-translational process that alters biological activity, solubility and lifetime of the glycoprotein in blood, and strongly depends on the type of cell and the cell culture conditions. A fast and simple method providing extensive carbohydrate information about the glycans present in rHuEPO and other glycoproteins is needed in order to improve current methods in drug development or product quality control. Here, an improved method for intact rHuEPO glycoform characterization by CZE-ESI-TOF MS has been developed using a novel capillary coating and compared to a previous study. Both methods allow a fast separation in combination with accurate mass characterization of the single protein isoforms. The novel dynamic coating provides a separation at an EOF close to zero, enabling better separation. This results in an improved mass spectrometric resolution and the detection of minor isoforms. In order to assign an unequivocal carbohydrate composition to every intact glycoform, a CZE-ESI-MS separation method for enzymatically released underivatized N-glycans has been developed. The TOF MS allows the correct identification of the glycans due to its high mass accuracy and resolution. Therefore, glycan modifications such as acetylation, oxidation, sulfation and even the exchange of OH by NH2 are successfully characterized. Information of the protein-backbone molecular mass has been combined with results from peptide analysis (revealing information about O-glycosylation) and from the glycan analysis, including the detection of as yet undescribed glycans containing four antennae and five sialic acids. This allows an unequivocal assignment of an overall glycosylation composition to the molecular masses obtained for the intact rHuEPO glycoforms.