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首页> 美国卫生研究院文献>Journal of Biomolecular Techniques : JBT >Characterization of Protein Glycosylation Using Chip-based Infusion Nanoelectrospray Linear Ion Trap Tandem Mass Spectrometry
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Characterization of Protein Glycosylation Using Chip-based Infusion Nanoelectrospray Linear Ion Trap Tandem Mass Spectrometry

机译：使用基于芯片的注入纳米电喷雾线性离子阱串联质谱法表征蛋白质糖基化

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摘要

Mass spectrometry (MS) has the potential to revolutionize structural glycobiology and help in the understanding of how post-translation events such as glycosylation affect protein activities. Several approaches to determine the structure of glycopeptides have been used successfully including fast atom bombardment, matrix-assisted laser desorption ionization, and electrospray ionization with a wide variety of mass analyzers. However, the identification of glycopeptides in a complex mixture still remains a challenge. The source of this challenge is primarily due to the poor ionization efficiency and rapid degradation of glycopeptides. In this report we describe the use of a chip-based infusion nanoelectrospray ionization technique in combination with a recently developed linear ion trap for identification and characterization of glycosylation in complex mixtures. Two standard synthetic glycans were analyzed using multiple-stage fragmentation analysis in both positive and negative ionization modes. In addition, the high mannose type N-glycosylation in ribonuclease B (RNase B) was used to map the glycosylation site and obtain the glycan structures. We were able to map the glycosylation site and obtain the glycan structures in RNase B in a single analysis. The results reported here demonstrate that the fully automated chip-based nanoelectrospray linear ion trap platform is a valuable system for oligosaccharide analyses due to the unique MS/MS and MSn capability of the linear ion trap and the extended analysis time provided by the ionization technique.
机译：质谱（MS）有可能彻底改变结构糖生物学，并有助于理解翻译后事件（例如糖基化）如何影响蛋白质活性。已经成功使用了几种确定糖肽结构的方法，包括快速原子轰击，基质辅助激光解吸电离和使用各种质量分析仪进行的电喷雾电离。然而，复杂混合物中糖肽的鉴定仍然是一个挑战。这一挑战的根源主要是由于离子化效率差和糖肽的快速降解。在本报告中，我们描述了基于芯片的注入式纳米电喷雾电离技术与最近开发的线性离子阱结合用于复杂混合物中糖基化的鉴定和表征。在正电离和负电离两种模式下，均采用多步碎裂分析法分析了两种标准合成聚糖。此外，核糖核酸酶B（RNase B）中的高甘露糖型N-糖基化用于定位糖基化位点并获得聚糖结构。我们能够在单个分析中定位糖基化位点并获得RNase B中的聚糖结构。此处报告的结果表明，基于线性离子阱的独特MS / MS和MS n 功能以及扩展的功能，基于芯片的全自动纳米电喷雾线性离子阱平台是一种低聚糖分析的有价值的系统电离技术提供的分析时间。

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