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连接位点与 从复杂的生物样品中辨别N-和O-聚糖。一个重大突破是， 该方法不需要截断聚糖，允许丰富的糖组学信息保持完整。 在分析期间。此外，这种称为同位素靶向糖蛋白组学（IsoTaG）的方法富集了 糖肽和允许前所未有的低丰度糖蛋白的检测。IsoTaG还减轻了 需要大量的分馏、质谱仪分析时间和计算时间。此外，自 我能够将IsoTag方法转移到没有糖生物学专业知识的感兴趣的实验室， 能够成功地对他们感兴趣的样品进行糖蛋白质组学分析。有了这些大的障碍 糖蛋白质组学的规模实施大大减少，我们在此提出使IsoTaG可获得 作为质谱分析（MS）核心设施提供的标准服务，向更广泛的科学界提供。 为了实现这一目标，我们将改进IsoTaG试剂并开发大规模合成， 分发给社区。我们还将标准化色谱法的各个方面， 分离糖肽，包括制作可用于校准保留时间的标准品。我们亦会 制定使用这些标准的协议，以调整仪器参数， 糖肽最后，我们将提供试剂和协议，以几个质谱核心设施 在仪器上试用IsoTag系统随后与设施的对话将有助于我们改善 方法，其目标是被具有高分辨率MS仪器的任何设施采用。

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.