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

7.项目摘要/摘要 我们最近发表了一种方法，首次允许多糖结构和附着位置 从复杂的生物样品中可以辨别出N-和O-多糖。一个重大突破是， 这种方法不需要截断糖链，从而使丰富的糖链信息保持不变 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，并允许前所未有的低丰度糖蛋白的检测。IsoTaG还可以缓解 需要大量的分馏、质谱仪分析时间和计算时间。此外，自那以后，我们已经 能够将IsoTag方法转移到之前没有糖生物学专业知识的感兴趣的实验室，他们是 能够成功地对他们感兴趣的样本进行糖蛋白质组分析。有了这些巨大的障碍 糖蛋白组学的规模化实施大大减少，我们在此建议使IsoTaG可访问 作为由质谱仪(MS)核心设施提供的标准服务提供给更广泛的科学界。 为了实现这一点，我们将改进IsoTaG试剂，并开发大规模合成以使 分发给社区。我们还将标准化层析的各个方面，用于 分离糖肽，包括制定可用于校准保留时间的标准。此外，我们还将 制定使用这些标准的协议来调整仪器参数，以实现最佳的碎裂 糖肽。最后，我们将向几个质谱学核心设施提供试剂和方案 在他们的仪器上试用IsoTag系统。随后与这些设施的对话将帮助我们改善 方法，其目标是它可以被任何具有高分辨率MS仪器的设施所采用。

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.