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

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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.