We have carefully selected two distinct sets of DBPs that drive most of the technical innovation outlined in this proposal. The first set of DBPs are focused on the development of specific tools to impact significantly externally funding research projects. The second set of DBPs are more focused on systems biology approaches to gain a better understanding of mammalian glycosylation and the role it plays in disease. These DBPs require robust datasets from a large collection of cell lineages and genetic perturbations in glycosylation machinery. Of course, these two sets of DBPs are also augmented by our multiple biomedical collaborations as well as our industry and academic mass spectrometry facility partners.
For the first set of investigators (Contessa, Dahms, Hart, Hoffmeister, Lau, and Wang), we specifically have chosen leaders and pioneers in their respective fields whose research endeavors are inhibited due to the lack of appropriate, robust, and/or high-throughput analytical techniques to address their biological questions and thus would significantly benefit from the development of particular new and improved tools to investigate glycosylation. We have brought in several investigators, including Hoffmeister, Lau, and Contessa, who will drive the development of our glycoprotein cell surface “capture and define” technology, including proteomics, site-mapping and direct glycopeptide analysis, and will benefit from our ability to make specific hES-derived cell lineages with specific glycosyltransferases knocked-out by CRISPR-Cas methodology. Likewise, we have partnered with several investigators, including Hart, Hoffmeister, and Lau, whose needs will drive our maturation of the static IDAWG approach for glycans and the need for developing our dynamic IDAWG approach for glycans and glycopeptides. We continue to partner with 2 existing DBPs (Dahms and Strauss) who will push our tool development with their needs for high-throughput, quantitative N- and O-glycan, as well as GSL, analyses. Wang as a DBP helps to drive the development of glycotranscriptomics and visualization tools. Along with these DBPs, several of our collaborators also help to drive the development of the tools and resources outlined in the TR&Ds.
For the second set of investigators (Aoki-Kinoshita, Lewis) we have chosen leaders in the fields of glycan pathway modeling and informatics analysis. In these cases the investigators are developing state-of-the-art approaches for modeling complex glycan pathway data, including pathway flux analysis, but have struggled to obtain appropriate data sets that can be used to test their modeling systems. The reporter glycoproteins expressed in pluripotent and differentiated hESC lines and in hESC lines harboring targeted glycogene knockouts will be analyzed as end-point glycosylated products in TR&D1. The resulting glycomic and transcriptomic profiles are ideal data sets for the type of modeling done in both the Aoki-Kinoshita and Lewis DBPs that use distinct approaches for pathway-based flux analysis. These DBPs also bridge to the technologies developed in TR&D3 for integrated pathway visualization. The Glycan Pathway Visualization tool will provide the ability to integrate pathway modeling output with comparative data from glycan and transcriptome profiling efforts. Interpretation of the integrated data in the context of a unified pathway diagram display framework will inform not only additional modeling efforts and optimization but also direct additional genetic perturbations of the reporter cell lines. Thus, an interactive and reciprocal set of driving interactions is anticipated with these DBPs. Additional challenges will be found in the DBP with Wang, where expansion of pathway visualization (TR&D 3) will be required for other glycan classes (proteoglycan biosynthesis) in order to integrate proteoglycan transcriptome profiling (TR&D1) with proteoglycan structural data generated in the Wang lab.