Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides
Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides
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DOI：
10.1002/pmic.200700968
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发表时间：
2008-07-01
期刊：
影响因子：
3.4
通讯作者：
Altmann, Friedrich
中科院分区：
文献类型：
--
作者：
Stadlmann, Johannes;Pabst, Martin;Altmann, Friedrich
本文介绍了两种分析抗体糖基化的LC-ESI-MS方法。在第一种方法中，用反相色谱分离胰酶糖肽，并用电喷雾质谱联用仪进行分析。这一“糖肽策略”允许对中性和唾液酸化的糖链结构进行蛋白质和亚类特异性的定量。可以从相同的数据中提取有关低糖或脱糖基化和蛋白质骨架(例如末端)的附加信息。在第二种LC-ESI-MS方法中，释放的低聚糖在多孔石墨炭(PGC)上被分离。因此，在一次层析运行中实现了抗体上中性和唾液酸化低聚糖的完整结构指定。这两种方法分别用于多克隆人免疫球蛋白、在CHO细胞(Rituximab、Xolair和Herceptin)、SP2/0(Erbitux和Remicade)或NS0细胞(Zenapax)中表达的商品化单抗，以及在CHO细胞或人细胞系中产生的抗HIV抗体4E10。这两种方法都需要相对较少的样品制备，并且可以应用于SDS-PAGE条带。这两种方法在峰分配的可靠性和未衍生多糖的MALDI-MS记录唾液酸化结构方面都优于非MS方法。对于快速而又详细的结构指认，石墨炭上的LC-MS取代了所有其他目前的方法。
Two LC-ESI-MS methods for the analysis of antibody glycosylation are presented. In the first approach, tryptic glycopeptides are separated by RP chromatography and analyzed by ESI-MS. This "glycopeptide strategy" allows a protein- and subclass-specific quantitation of both neutral and sialylated glycan structures. Additional information about under- or deglycosylation and the protein backbone, e.g., termini, can be extracted from the same data. In the second LC-ESI-MS method, released oligosaccharides are separated on porous graphitic carbon (PGC). A complete structural assignment of neutral and sialylated oligosaccharides occurring on antibodies is thereby achieved in one chromatographic run. The two methods were applied to polyclonal human IgG, to commercial mAb expressed in CHO cells (Rituximab, Xolair, and Herceptin), in SP2/0 (Erbitux and Remicade) or NS0 cells (Zenapax) and the anti-HIV antibody 4E10 produced either in CHO cells or in a human cell line. Both methods require comparably little sample preparation and can be applied to SDS-PAGE bands. They both outperform non-MS methods in terms of reliability of peak assignment and MALDI-MS of underivatized glycans with regard to the recording of sialylated structures. Regarding fast and yet detailed structural assignment, LC-MS on graphitic carbon supersedes all other current methods.