John W. Thompson

All Publications

• Functional analysis of O-GlcNAcylation by networking of OGT interactors and substrates. Nature chemical biology Griffin, M. E., Thompson, J. W., Xiao, Y., Sweredoski, M. J., Jensen, E. H., Aksenfeld, R. B., Awad, H., Kim, T. D., Schacht, A. L., Choudhry, P., Koldobskaya, Y., Lomenick, B., Garbis, S. D., Moradian, A., Hsieh-Wilson, L. C. 2026

  Abstract

  The post-translational modification (PTM) of proteins by O-linked β-N-acetyl-D-glucosamine (O-GlcNAcylation) is widely found across the proteome and regulates diverse cellular processes, from transcription and translation to signal transduction and metabolism. However, most functional studies to date have focused on individual modifications, overlooking other simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here we describe networking of O-GlcNAc transferase interactors and substrates (NOTISE), a systems-level approach that monitors O-GlcNAcylation rapidly and comprehensively across the proteome to reveal important functional and regulatory relationships. The NOTISE method integrates affinity purification-mass spectrometry and site-specific chemoproteomic technologies with network generation to connect putative upstream regulators and downstream targets of O-GlcNAcylation. The resulting data-rich networks identify critical conserved activities of O-GlcNAcylation and tissue-specific functions. This holistic and unbiased approach provides a broadly applicable framework to catalyze investigations into the functional roles of coordinated, multisubstrate PTMs in specific cellular and physiological contexts.

  View details for DOI 10.1038/s41589-025-02108-7

  View details for PubMedID 41634442

  View details for PubMedCentralID 5667541

• Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting. Biochemistry Darabedian, N., Thompson, J. W., Chuh, K. N., Hsieh-Wilson, L. C., Pratt, M. R. 2018; 57 (40): 5769-5774

  Abstract

  The dynamic modification of intracellular proteins by O-linked β -N-acetylglucosamine (O-GlcNAcylation) plays critical roles in many cellular processes. Although various methods have been developed for O-GlcNAc detection, there are few techniques for monitoring glycosylation stoichiometry and state (i.e., mono-, di-, etc., O-GlcNAcylated). Measuring the levels of O-GlcNAcylation on a given substrate protein is important for understanding the biology of this critical modification and for prioritizing substrates for functional studies. One powerful solution to this limitation involves the chemoenzymatic installation of polyethylene glycol polymers of defined molecular mass onto O-GlcNAcylated proteins. These "mass tags" produce shifts in protein migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that can be detected by Western blotting. Broad adoption of this method by the scientific community has been limited, however, by a lack of commercially available reagents and well-defined protein standards. Here, we develop a "click chemistry" approach to this method using entirely commercial reagents and confirm the accuracy of the approach using a semisynthetic O-GlcNAcylated protein. Our studies establish a new, expedited experimental workflow and standardized methods that can be readily utilized by non-experts to quantify the O-GlcNAc stoichiometry and state on endogenous proteins in any cell or tissue lysate.

  View details for DOI 10.1021/acs.biochem.8b00648

  View details for PubMedID 30169966

  View details for PubMedCentralID PMC6211186

• Methods for the Detection, Study, and Dynamic Profiling of O-GlcNAc Glycosylation. Methods in enzymology Thompson, J. W., Griffin, M. E., Hsieh-Wilson, L. C. 2018; 598: 101-135

  Abstract

  The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. In this chapter, we describe a set of chemoenzymatic labeling methods to (1) detect O-GlcNAcylation on proteins of interest, (2) monitor changes in both the total levels of O-GlcNAcylation and its stoichiometry on proteins of interest, and (3) enable mapping of O-GlcNAc to specific serine/threonine residues within proteins to facilitate functional studies. First, we outline a procedure for the expression and purification of a multiuse mutant galactosyltransferase enzyme (Y289L GalT). We then describe the use of Y289L GalT to modify O-GlcNAc residues with a functional handle, N-azidoacetylgalactosamine (GalNAz). Finally, we discuss several applications of the copper-catalyzed azide-alkyne cycloaddition "click" reaction to attach various alkyne-containing chemical probes to GalNAz and demonstrate how this functionalization of O-GlcNAc-modified proteins can be used to realize (1)-(3) above. Overall, these methods, which utilize commercially available reagents and standard protein analytical tools, will serve to advance our understanding of the diverse and important functions of O-GlcNAcylation.

  View details for DOI 10.1016/bs.mie.2017.06.009

  View details for PubMedID 29306432

  View details for PubMedCentralID PMC5886303

• Deciphering the Functions of O-GlcNAc Glycosylation in the Brain: The Role of Site-Specific Quantitative O-GlcNAcomics. Biochemistry Thompson, J. W., Sorum, A. W., Hsieh-Wilson, L. C. 2018; 57 (27): 4010-4018

  Abstract

  The dynamic posttranslational modification O-linked β- N-acetylglucosamine glycosylation (O-GlcNAcylation) is present on thousands of intracellular proteins in the brain. Like phosphorylation, O-GlcNAcylation is inducible and plays important functional roles in both physiology and disease. Recent advances in mass spectrometry (MS) and bioconjugation methods are now enabling the mapping of O-GlcNAcylation events to individual sites in proteins. However, our understanding of which glycosylation events are necessary for regulating protein function and controlling specific processes, phenotypes, or diseases remains in its infancy. Given the sheer number of O-GlcNAc sites, methods for identifying promising sites and prioritizing them for time- and resource-intensive functional studies are greatly needed. Revealing sites that are dynamically altered by different stimuli or disease states will likely go a long way in this regard. Here, we describe advanced methods for identifying O-GlcNAc sites on individual proteins and across the proteome and for determining their stoichiometry in vivo. We also highlight emerging technologies for quantitative, site-specific MS-based O-GlcNAc proteomics (O-GlcNAcomics), which allow proteome-wide tracking of O-GlcNAcylation dynamics at individual sites. These cutting-edge technologies are beginning to bridge the gap between the high-throughput cataloguing of O-GlcNAcylated proteins and the relatively low-throughput study of individual proteins. By uncovering the O-GlcNAcylation events that change in specific physiological and disease contexts, these new approaches are providing key insights into the regulatory functions of O-GlcNAc in the brain, including their roles in neuroprotection, neuronal signaling, learning and memory, and neurodegenerative diseases.

  View details for DOI 10.1021/acs.biochem.8b00516

  View details for PubMedID 29936833

  View details for PubMedCentralID PMC6058732

• Activities for Middle School Students To Sleuth a Chemistry "Whodunit" and Investigate the Scientific Method JOURNAL OF CHEMICAL EDUCATION Meyer, A. F., Knutson, C. M., Finkenstaedt-Quinn, S. A., Gruba, S. M., Meyer, B. M., Thompson, J. W., Maurer-Jones, M. A., Halderman, S., Tillman, A. S., DeStefano, L., Haynes, C. L. 2014; 91 (3): 410-413

  View details for DOI 10.1021/ed4006562

  View details for Web of Science ID 000333319500017

• Time- and concentration-dependent effects of exogenous serotonin and inflammatory cytokines on mast cell function. ACS chemical biology Gruba, S. M., Meyer, A. F., Manning, B. M., Wang, Y., Thompson, J. W., Dalluge, J. J., Haynes, C. L. 2014; 9 (2): 503-9

  Abstract

  Mast cells play a significant role in both the innate and adaptive immune response; however, the tissue-bound nature of mast cells presents an experimental roadblock to performing physiologically relevant mast cell experiments. In this work, a heterogeneous cell culture containing primary culture murine peritoneal mast cells (MPMCs) was studied to characterize the time-dependence of mast cell response to allergen stimulation and the time- and concentration-dependence of the ability of the heterogeneous MPMC culture to uptake and degranulate exogenous serotonin using high performance liquid chromatography (HPLC) coupled to an electrochemical detector. Additionally, because mast cells play a central role in asthma, MPMCs were exposed to CXCL10 and CCL5, two important asthma-related inflammatory cytokines that have recently been shown to induce mast cell degranulation. MPMC response to both allergen exposure and cytokine exposure was evaluated for 5-HT secretion and bioactive lipid formation using ultraperformance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer (UPLC-MS/MS). In this work, MPMC response was shown to be highly regulated and responsive to subtle alterations in a complex environment through time- and concentration-dependent degranulation and bioactive lipid formation. These results highlight the importance of selecting an appropriate mast cell model when studying mast cell involvement in allergic response and inflammation.

  View details for DOI 10.1021/cb400787s

  View details for PubMedID 24304209

  View details for PubMedCentralID PMC4083829

• Isotope-dilution UPLC-MS/MS determination of cell-secreted bioactive lipids. The Analyst Meyer, A. F., Thompson, J. W., Wang, Y., Koseoglu, S., Dalluge, J. J., Haynes, C. L. 2013; 138 (19): 5697-705

  Abstract

  Secreted bioactive lipids play critical roles in cell-to-cell communication and have been implicated in inflammatory immune responses such as anaphylaxis, vasodilation, and bronchoconstriction. Analysis of secreted bioactive lipids can be challenging due to their relatively short lifetimes and structural diversity. Herein, a method has been developed using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to quantify five cell-secreted, structurally and functionally diverse bioactive lipids (PGD2, LTC4, LTD4, LTE4, PAF) that play roles in inflammation. Sample analysis time is 5 min, and isotopically labeled internal standards are used for quantification. This method was applied to an immortal secretory cell line (RBL-2H3), a heterogeneous primary cell culture containing peritoneal mast cells, and murine platelets. In RBL cell supernatant samples, intrasample precisions ranged from 7.32-21.6%, averaging 17.0%, and spike recoveries in cell supernatant matrices ranged from 88.0-107%, averaging 97.0%. Calibration curves were linear from 10 ng mL(-1) to 250 ng mL(-1), and limits of detection ranged from 0.0348 ng mL(-1) to 0.803 ng mL(-1). This method was applied to the determination of lipid secretion from mast cells and platelets, demonstrating broad applicability for lipid measurement in primary culture biological systems.

  View details for DOI 10.1039/c3an00875d

  View details for PubMedID 23923125

• Development of screening assays for nanoparticle toxicity assessment in human blood: preliminary studies with charged Au nanoparticles. Nanomedicine (London, England) Love, S. A., Thompson, J. W., Haynes, C. L. 2012; 7 (9): 1355-64

  Abstract

  As nanoparticles have found increased use in both consumer and medical applications, corresponding increases in possible exposure to humans necessitate studies examining the impacts of these nanomaterials in biological systems. This article examines the effects of approximately 30-nm-diameter gold nanoparticles, with positively and negatively charged surface coatings in human blood. Here, we study the exposure effects, with up to 72 h of exposure to 5, 15, 25 and 50 µg/ml nanoparticles on hemolysis, reactive oxygen species (ROS) generation and platelet aggregation in subsets of cells from human blood. Assessing viability with hemolysis, results show significant changes in a concentration-dependent fashion. Rates of ROS generation were investigated using the dichlorofluorscein diacetate-based assay as ROS generation is a commonly suspected mechanism of nanoparticle toxicity; herein, ROS was not a significant factor. Optical monitoring of platelet aggregation revealed that none of the examined nanoparticles induced aggregation upon short-term exposure.

  View details for DOI 10.2217/nnm.12.17

  View details for PubMedID 22583573

• Assessing nanoparticle toxicity. Annual review of analytical chemistry (Palo Alto, Calif.) Love, S. A., Maurer-Jones, M. A., Thompson, J. W., Lin, Y. S., Haynes, C. L. 2012; 5: 181-205

  Abstract

  Nanoparticle toxicology, an emergent field, works toward establishing the hazard of nanoparticles, and therefore their potential risk, in light of the increased use and likelihood of exposure. Analytical chemists can provide an essential tool kit for the advancement of this field by exploiting expertise in sample complexity and preparation as well as method and technology development. Herein, we discuss experimental considerations for performing in vitro nanoparticle toxicity studies, with a focus on nanoparticle characterization, relevant model cell systems, and toxicity assay choices. Additionally, we present three case studies (of silver, titanium dioxide, and carbon nanotube toxicity) to highlight the important toxicological considerations of these commonly used nanoparticles.

  View details for DOI 10.1146/annurev-anchem-062011-143134

  View details for PubMedID 22524221