Analysis of protein glycosylation by mass spectrometry
Analysis of protein glycosylation by mass spectrometry
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DOI：
10.1007/bf02745880
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发表时间：
1994-01-01
影响因子：
2.6
通讯作者：
Nilsson, Bo
中科院分区：
文献类型：
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作者：
Nilsson, Bo
用于治疗和诊断的基于蛋白质的药物的制药市场不断增长。分子生物学和细胞生物学的快速发展导致了用于生产重组蛋白和单克隆抗体的表达系统的产生。当在具有糖基化能力的细胞系统中表达时，这些蛋白质被糖基化。对于用于治疗性给药的糖蛋白，重要的是了解碳水化合物侧链的结构，以避免产生结构的细胞系统，所述结构在人体中可引起不期望的反应，例如，免疫学和不利血清清除率。糖蛋白寡糖的结构分析需要复杂的仪器，如质谱仪和核磁共振光谱仪。然而，在进行结构分析之前，必须将碳水化合物链从蛋白质中释放出来并纯化至均一，这通常是最耗时的步骤。质谱技术在蛋白质糖基化分析中一直发挥着重要作用。与其他光谱方法相比，其上级灵敏度是其主要优点。然而，由于组成单糖残基的化学性质，碳水化合物的结构分析面临几个问题。对于寡糖或糖缀合物，来自质谱的结构信息基本上限于单糖序列、分子量，并且仅在例外情况下可以获得糖苷键位置。为了完全建立寡糖结构，必须确定几个其他结构参数，例如，连接位置、异头构型和单糖结构单元的鉴定。解决这些问题的一种方法是对糖缀合物进行化学预处理，以特异性地修饰碳水化合物链。为了引入特定的修饰，我们使用了高碘酸盐氧化和三氟乙酸解，目的是通过质谱法确定糖苷键的位置。
There is a growing pharmaceutical market for protein-based drugs for use in therapy and diagnosis. The rapid developments in molecular and cell biology have resulted in production of expression systems for manufacturing of recombinant proteins and monoclonal antibodies. These proteins are glycosylated when expressed in cell systems with glycosylation ability. For glycoproteins intended for therapeutic administration it is important to have knowledge about the structure of the carbohydrate side chains to avoid cell systems that produce structures, which in humans can cause undesired reactions, e.g., immunological and unfavorable serum clearance rate. Structural analysis of glycoprotein oligosaccharides requires sophisticated instruments like mass spectrometers and nuclear magnetic resonance spectrometers. However, before the structural analysis can be conducted, the carbohydrate chains have to be released from the protein and purified to homogeneity, and this is often the most time-consuming step. Mass spectrometry has played and still plays an important role in analysis of protein glycosylation. The superior sensitivity compared to other spectroscopic methods is its main asset. Structural analysis of carbohydrates faces several problems, however, due to the chemical nature of the constituent monosaccharide residues. For oligosaccharides or glycoconjugates, the structural information from mass spectrometry is essentially limited to monosaccharide sequence, molecular weight, and only in exceptional cases glycosidic linkage positions can be obtained. In order to completely establish an oligosaccharide structure, several other structural parameters have to be determined, e.g., linkage positions, anomeric configuration and identification of the monosaccharide building blocks. One way to address some of these problems is to work on chemical pretreatment of the glycoconjugate, to specifically modify the carbohydrate chain. In order to introduce specific modifications, we have used periodate oxidation and trifluoroacetolysis with the objective of determining glycosidic linkage positions by mass spectrometry.