Functional glycoproteomics by integrated network assembly and partitioning
Creators
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1.
California Institute of Technology
Abstract
The post-translational modification (PTM) of proteins by O-linked β-N-acetyl-D-glucosamine (O-GlcNAcylation) is widespread across the proteome during the lifespan of all multicellular organisms. However, nearly all functional studies have focused on individual protein modifications, overlooking the multitude of simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here, we describe Networking of Interactors and SubstratEs (NISE), a novel, systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the proteome. Our method integrates affinity purification-mass spectrometry (AP-MS) and site-specific chemoproteomic technologies with network generation and unsupervised partitioning to connect potential upstream regulators with downstream targets of O-GlcNAcylation. The resulting network provides a data-rich framework that reveals both conserved activities of O-GlcNAcylation such as epigenetic regulation as well as tissue-specific functions like synaptic morphology. Beyond O-GlcNAc, this holistic and unbiased systems-level approach provides a broadly applicable framework to study PTMs and discover their diverse roles in specific cell types and biological states.
Copyright and License
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Acknowledgement
We thank S. Pease and staff at the Caltech Genetically Engineered Mouse Services Core for help with generating the OGT-FH mouse line. This project was supported by the National Institutes of Health (RF1AG060540 to L.C.H.-W. and T32GM008042, T32GM007616, and F30AG055314 to J.W.T), UCLA-Caltech Medical Scientist Training Program (J.W.T), National Science Foundation (GRFP DGE-1144469 to M.E.G.), and Department of Defense (NDSEG to E.H.J.).
Contributions
Conceptualization: M.E.G., J.W.T., Y.X., and L.C.H.-W.; Methodology: M.E.G., J.W.T., Y.X., and M.J.S.; Software: J.W.T., M.J.S., and P.C.; Investigation: M.E.G., J.W.T., Y.X., R.B.A., E.H.J., Y.K., A.L.S., T.D.K., A.M., and B.L.; Writing – Original Draft: M.E.G., J.W.T., Y.X., and L.C.H.-W.; Writing – Review & Editing: M.E.G., J.W.T., and L.C.H.-W.; Visualization: M.E.G., J.W.T. and L.C.H.-W.; Supervision: M.E.G., S.D.G., and L.C.H.-W.; Funding Acquisition: L.C.H.-W.
Conflict of Interest
S.D.G. is founder and CEO/CTO of Proteas Bioanalytics, Inc.
Files
2023.06.13.541482v1.full.pdf
Additional details
Identifiers
- PMCID
- PMC10312638
Funding
- National Institutes of Health
- RF1AG060540
- National Institutes of Health
- NIH Predoctoral Fellowship T32GM008042
- National Institutes of Health
- T32GM007616
- National Institutes of Health
- NIH Postdoctoral Fellowship F30AG055314
- California Institute of Technology
- UCLA-Caltech Medical Scientist Training Program
- National Science Foundation
- NSF Graduate Research Fellowship DGE-1144469
- United States Department of Defense
- National Defense Science and Engineering Graduate (NDSEG) Fellowship