Making glycoproteomics via mass spectrometry more accessible to the greater scientific community

7. 项目概要/摘要 我们最近发表了一种方法，首次允许聚糖结构和附着位点 可以从复杂的生物样品中辨别出 N- 和 O- 聚糖。一个重大突破是 该方法不需要截断聚糖，从而使丰富的糖组信息保持完整 在分析过程中。此外，这种称为同位素靶向糖蛋白组学 (IsoTaG) 的方法丰富了 糖肽并允许前所未有地检测低丰度糖蛋白。 IsoTaG 还可以减轻 需要大量的分馏、质谱仪分析时间和计算时间。另外，我们从那时起 能够将 IsoTag 方法转移到以前没有糖生物学专业知识的感兴趣的实验室，这些实验室 能够成功地对其感兴趣的样品进行糖蛋白组分析。有了这些大的障碍 糖蛋白组学的规模实施大大减少，我们在此建议使 IsoTaG 易于使用 作为质谱 (MS) 核心设施提供的标准服务，向更广泛的科学界提供服务。 为了实现这一目标，我们将改进 IsoTaG 试剂并开发大规模合成方法，以实现 分发给社区。我们还将标准化用于色谱的各个方面 分离糖肽，包括制作可用于校准保留时间的标准品。此外，我们还将 开发使用这些标准的协议来调整仪器参数，以实现最佳的碎片化 糖肽。最后，我们将为多个质谱核心设施提供试剂和方案 在他们的仪器上试用 IsoTag 系统。随后与设施的对话将帮助我们改进 方法的目标是任何拥有高分辨率 MS 仪器的设施都可以采用该方法。

7. PROJECT SUMMARY/ABSTRACT We very recently published a method that, for the first time, allows glycan structures and sites of attachment to be discerned from complex biological samples for both N- and O-glycans alike. A major breakthrough is that this method does not require truncation of the glycans, allowing the rich glycomic information to remain intact during analysis. In addition, this method, termed Isotope-Targeted Glycoproteomics (IsoTaG) enriches glycopeptides and allows unprecedented detection of low abundance glycoproteins. IsoTaG also mitigates the need for extensive fractionation, mass spectrometer analysis time, and computation time. Also, we have since been able to transfer the IsoTag method to interested labs that have no prior glycobiology expertise, who were able to successfully perform glycoproteomic analysis on their samples of interest. With these barriers to large scale implementation of glycoproteomics substantially reduced, we propose herein to make IsoTaG accessible to the broader scientific community as a standard service offered by mass spectrometry (MS) core facilities. To accomplish this, we will improve the IsoTaG reagent and develop a large scale synthesis to enable distribution to the community. We will also standardize various aspects of the chromatography used to separate glycopeptides, including making standards that can be used to calibrate retention times. Also, we will develop protocols for use of these standards to tune instrument parameters for optimal fragmentation of the glycopeptides. Finally, we will provide the reagents and protocols to several mass spectrometry core facilities to trial use of the IsoTag system on their instruments. Ensuing dialog with the facilities will help us improve the method with the goal of it being adoptable by any facility that has high resolution MS instrumentation.