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.
Most human proteins are decorated with sugars, which is why we call them 'glycoproteins'. The shapes, sizes and attachment sites of the sugars change when the cell making the glycoprotein transforms from healthy to diseased, but these can only be observed currently by scientific labs that specialize in this area and own very expensive equipment. The goal of this project is to develop standard technologies that can be used by the broad scientific community for detecting changes in glycoproteins, which will aid in medical investigations such as cancer research and diagnosis down the line.
| Spiciarich, David R; Oh, Stephen T; Foley, Amy et al. (2018) A Novel Germline Variant in CSF3R Reduces N-Glycosylation and Exerts Potent Oncogenic Effects in Leukemia. Cancer Res 78:6762-6770 |
| Woo, Christina M; Lund, Peder J; Huang, Andrew C et al. (2018) Mapping and Quantification of Over 2000 O-linked Glycopeptides in Activated Human T Cells with Isotope-Targeted Glycoproteomics (Isotag). Mol Cell Proteomics 17:764-775 |
| Woo, Christina M; Felix, Alejandra; Byrd, William E et al. (2017) Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes. J Proteome Res 16:1706-1718 |
| Spiciarich, David R; Nolley, Rosalie; Maund, Sophia L et al. (2017) Bioorthogonal Labeling of Human Prostate Cancer Tissue Slice Cultures for Glycoproteomics. Angew Chem Int Ed Engl 56:8992-8997 |
