Glycan Labeling Research Articles

Deciphering the Cell Surface Sugar-Coating via Biochemical Pathways.

Cell surface components, specifically glycans, play a significant role in several biological functions like cell structure, crosstalk between cells, and eventual target recognition of the cells for therapeutics. The dense layer of glycans, i. e., glycocalyx, could differ in taxon, species, and cell type. Glycans are coupled with lipids and proteins to form glycolipids, glycoproteins, proteoglycans, and glycosylphosphatidylinositol-anchored proteins, making their study challenging. However, understanding glycosylation at the cellular level is vital for fundamental research and advancing glycan-targeted therapy. Among different pathways, metabolic glycan labelling uses the natural metabolic processes of the cell to introduce abiotic functionality into glycan residues. The Bertozzi group pioneered metabolic oligosaccharide engineering using glycan salvage pathways to convert monosaccharides with unnatural modifications. This eventually results in the probe becoming part of the complex cellular glycan structures via click chemistry using copper. On the other hand, the boronic acid-based probe can recognise carbohydrates in a single step without any chemical modification of the surface. This review discusses the significance of glycans as biomarkers for different diseases and the necessity to evaluate them in situ within the physiological environment. The review also discusses the prospect of this field and its potential applications.

Chemical Recording of Pump-Specific Drug Efflux in Living Cells.

Drug efflux-a process primarily facilitated by efflux pumps such as multidrug resistance proteins (MRPs)-plays a pivotal role in cellular resistance to chemotherapies. Conventional approaches to assess drug efflux are predominantly conducted in vitro and often lack pump specificity. Here we report the bioorthogonal reporter inhibiting efflux (BRIEF) strategy, which enables the recording of pump-specific drug efflux in living cells. In BRIEF, a specific substrate is engineered as a bioorthogonal efflux probe (BEP) for specific pumps. The cellular concentration and protein labeling level of the probe can be augmented when the test drug is transported by the same pumps. Serendipitously, we discovered that per-O-acetylated unnatural monosaccharides, initially designed for metabolic glycan labeling, are exported by some MRPs. Using Ac4GlcNAl as a BEP, we studied the structure-efflux relationship of flavonoids and identified small molecules, including tannic acid, cholesterol and gallic acid, as novel MRP substrates in high-throughput screening. Tannic acid, known for anti-tumor and anti-SARS-CoV-2 properties, showed increased efficacy upon MRP inhibition. Additionally, BRIEF was adapted to assess p-glycoprotein-mediated efflux using Rhodamine 123 as a BEP, leveraging its light-activatable proximity labeling ability. BRIEF provides a versatile approach to investigate drug efflux and enhance chemotherapy strategies.

Fast Metabolic Glycan Labeling for Click-Labeling and Imaging of Sialoglycans in Living Acute Brain Slices from Mice and Human Patients.

Living acute brain slices provide a practical platform for imaging sialylation in human brain pathology. However, the limited lifespan of acute brain slices has impeded the use of metabolic glycan labeling (MGL), which requires long-term incubation of clickable unnatural sugars such as N-azidoacetylmannosamine (ManNAz) to metabolically incorporate azides into sialoglycans. Here, we report a fast variant of MGL (fMGL), in which ManNAz-6-phosphate enables efficient azidosugar incorporation within 12 h by bypassing the bottleneck step in the sialic acid biosynthesis pathway, followed by click-labeling with fluorophores and imaging of sialoglycans in acute brain slices from mice and human patients. In the clinical samples of ganglioglioma, fMGL-based imaging reveals specific upregulation of sialylation in astrocyte-like but not neuron-like tumor cells. In addition, fMGL is integrated with click-expansion microscopy for high-resolution imaging of sialoglycans in brain slices. The fMGL strategy should find broad applications in the tissue imaging of glycans and surgical pathology.

Adipose microenvironment promotes hypersialylation of ovarian cancer cells.

Ovarian and other peritoneal cancers have a strong tendency to metastasize into the surrounding adipose tissue. This study describes an effect of the adipose microenvironment on upregulation of sialic acid-containing glycans in ovarian cancer (OC). Heterogeneous populations of glycosylated OC tumors converged to a highly sialylated cell state that regulates tumorigenesis in an immune-dependent manner. We modeled the adipose microenvironment by conditioning growth media with human patient-derived adipose tissue. OC cell lines grown in the presence vs. absence of adipose conditioned media (ACM) were characterized by transcriptomics, western blotting, and chemical biology glycan labeling methods. Fluorescence-activated cell sorting was used to separate adipose-driven upregulation of hypersialylated ("SNA-high") vs. hyposialylated ("SNA-low") OC subpopulations. The two subpopulations were characterized by further transcriptomic and quantitative polymerase chain reaction analyses, then injected into a syngeneic mouse model. Immune system involvement was implicated using wild type and athymic nude mice with a primary endpoint of overall survival. Adipose conditioning resulted in upregulation of sialyltransferases ST3GAL1, ST6GAL1, ST6GALNAC3, and ST8Sia1. In culture, OC cells displayed two distinct sialylated subpopulations that were stable for up to 9 passages, suggesting inherent heterogeneity in sialylation that is maintained throughout cell division and media changes. OC tumors that implanted in the omental adipose tissue exclusively reprogrammed to the highly sialylated subpopulation. In wild type C57BL/6 mice, only the hypersialylated SNA-high subpopulation implanted in the adipose, whereas the hyposialylated SNA-low subpopulation failed to be tumorigenic (p=0.023, n=5). In the single case where SNA-low established a tumor, post-mortem analysis revealed reprogramming of the tumor to the SNA-high state in vivo. In athymic nude mice, both subpopulations rapidly formed tumors, implicating a role of the adaptive immune system. These findings suggest a model of glycan-dependent tumor evolution wherein the adipose microenvironment reprograms OC to a tumorigenic state that resists the adaptive immune system. Mechanistically, adipose factors upregulate sialyltransferases. To our knowledge, this is the first demonstration of the effect of adipose microenvironment on OC tumor sialylation. Our results set the stage for translational applications targeting sialic acid pathways in OC and other peritoneal cancer tumorigenesis and metastasis.

Glycan clock of ageing-analytical precision and time-dependent inter- and i-individual variability.

Ageing is a complex biological process with variations among individuals, leading to the development of ageing clocks to estimate biological age. Glycans, particularly in immunoglobulin G (IgG), have emerged as potential biomarkers of ageing, with changes in glycosylation patterns correlating with chronological age.For precision analysis, three different plasma pools were analysed over 26days in tetraplicates, 312 samples in total. In short-term variability analysis, two cohorts were analysed: AstraZeneca MFO cohort of 26 healthy individuals (median age 20) and a cohort of 70 premenopausal Chinese women (median age 22.5) cohort monitored over 3months. Long-term variability analysis involved two adult men aged 47 and 57, monitored for 5 and 10years, respectively. Samples were collected every 3months and 3weeks, respectively. IgG N-glycan analysis followed a standardized approach by isolating IgG, its subsequent denaturation and deglycosylation followed by glycan cleanup and labelling. Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ultra-performance liquid chromatography analyses were employed for glycan profiling. Statistical analysis involved normalization, batch correction, and linear mixed models to assess time effects on derived glycan traits.The intermediate precision results consistently exhibited very low coefficient of variation values across all three test samples. This consistent pattern underscores the high level of precision inherent in the CGE method for analysing the glycan clock of ageing. The AstraZeneca MFO cohort did not show any statistically significant trends, whereas the menstrual cycle cohort exhibited statistically significant trends in digalactosylated (G2), agalactosylated (G0) and fucosylation (F). These trends were attributed to the effects of the menstrual cycle. Long-term stability analysis identified enduring age-related trends in both subjects, showing a positive time effect in G0 and bisected N-acetylglucosamine, as well as a negative time effect in G2 and sialylation, aligning with earlier findings. Time effects measured for monogalactosylation, and F remained substantially lower than ones observed for other traits.The study found that IgG N-glycome analysis using CGE-LIF exhibited remarkably high intermediate precision. Moreover, the study highlights the short- and long-term stability of IgG glycome composition, coupled with a notable capacity to adapt and respond to physiological changes and environmental influences such as hormonal changes, disease, and interventions. The discoveries from this study propel personalized medicine forward by deepening our understanding of how IgG glycome relates to age-related health concerns. This study underscores the reliability of glycans as a biomarker for tracking age-related changes and individual health paths.

Tumor-Targeted Oxaliplatin(IV) Prodrug Delivery Based on ROS-Regulated Cancer-Selective Glycan Labeling.

Platinum-drug-based chemotherapy in clinics has achieved great success in clinical malignancy therapy. However, unpredictable off-target toxicity and the resulting severe side effects in the treatment are still unsolved problems. Although metabolic glycan labeling-mediated tumor-targeted therapy has been widely reported, less selective metabolic labeling in vivo limited its wide application. Herein, a novel probe of B-Ac3ManNAz that is regulated by reactive oxygen species in tumor cells is introduced to enhance the recognition and cytotoxicity of DBCO-modified oxaliplatin(IV) via bioorthogonal chemistry. B-Ac3ManNAz was synthesized from Ac4ManNAz by incorporation with 4-(hydroxymethyl) benzeneboronic acid pinacol ester (HBAPE) at the anomeric position, which is confirmed to be regulated by ROS and could robustly label glycans on the cell surface. Moreover, N3-treated tumor cells could enhance the tumor accumulation of DBCO-modified oxaliplatin(IV) via click chemistry meanwhile reduce the off-target distribution in normal tissue. Our strategy provides an effective metabolic precursor for tumor-specific labeling and targeted cancer therapies.

Native N-glycome profiling of single cells and ng-level blood isolates using label-free capillary electrophoresis-mass spectrometry

The development of reliable single-cell dispensers and substantial sensitivity improvement in mass spectrometry made proteomic profiling of individual cells achievable. Yet, there are no established methods for single-cell glycome analysis due to the inability to amplify glycans and sample losses associated with sample processing and glycan labeling. In this work, we present an integrated platform coupling online in-capillary sample processing with high-sensitivity label-free capillary electrophoresis-mass spectrometry for N-glycan profiling of single mammalian cells. Direct and unbiased quantitative characterization of single-cell surface N-glycomes are demonstrated for HeLa and U87 cells, with the detection of up to 100 N-glycans per single cell. Interestingly, N-glycome alterations are unequivocally detected at the single-cell level in HeLa and U87 cells stimulated with lipopolysaccharide. The developed workflow is also applied to the profiling of ng-level amounts (5–500 ng) of blood-derived protein, extracellular vesicle, and total plasma isolates, resulting in over 170, 220, and 370 quantitated N-glycans, respectively.

In-Membrane Enrichment and Peptic Digestion to Facilitate Analysis of Monoclonal Antibody Glycosylation.

The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the glycosylation profile of mAbs during production is essential to ensuring their safety and efficacy. This research aims to rapidly isolate and digest mAbs for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of glycans and monitoring of glycosylation patterns, potentially during manufacturing. Immobilization of an Fc region-specific ligand, oFc20, in a porous membrane enables the enrichment of mAbs from cell culture supernatant and efficient elution with an acidic solution. Subsequent digestion of the mAb eluate occurred in a pepsin-modified membrane within 5 min. The procedure does not require alkylation and desalting, greatly shortening the sample preparation time. Subsequent LC-MS/MS analysis identified 11 major mAb N-glycan proteoforms and assessed the relative peak areas of the glycosylated peptides. This approach is suitable for the glycosylation profiling of various human IgG mAbs, including biosimilars and different IgG subclasses. The total time required for this workflow is less than 2 h, whereas the conventional enzymatic release and labeling of glycans can take much longer. Thus, the integrated membranes are suitable for facilitating the analysis of mAb glycosylation patterns.

Engineered Cell Membrane-Camouflaged Nanomaterials for Biomedical Applications.

Recent strides in nanomaterials science have paved the way for the creation of reliable, effective, highly accurate, and user-friendly biomedical systems. Pioneering the integration of natural cell membranes into sophisticated nanocarrier architectures, cell membrane camouflage has emerged as a transformative approach for regulated drug delivery, offering the benefits of minimal immunogenicity coupled with active targeting capabilities. Nevertheless, the utility of nanomaterials with such camouflage is curtailed by challenges like suboptimal targeting precision and lackluster therapeutic efficacy. Tailored cell membrane engineering stands at the forefront of biomedicine, equipping nanoplatforms with the capacity to conduct more complex operations. This review commences with an examination of prevailing methodologies in cell membrane engineering, spotlighting strategies such as direct chemical modification, lipid insertion, membrane hybridization, metabolic glycan labeling, and genetic engineering. Following this, an evaluation of the unique attributes of various nanomaterials is presented, delivering an in-depth scrutiny of the substantial advancements and applications driven by cutting-edge engineered cell membrane camouflage. The discourse culminates by recapitulating the salient influence of engineered cell membrane camouflage within nanomaterial applications and prognosticates its seminal role in transformative healthcare technologies. It is envisaged that the insights offered herein will catalyze novel avenues for the innovation and refinement of engineered cell membrane camouflaged nanotechnologies.

Anastrozole and Tamoxifen Impact on IgG Glycome Composition Dynamics in Luminal A and Luminal B Breast Cancers.

This study examines the intricate relationship between protein glycosylation dynamics and therapeutic responses in Luminal A and Luminal B breast cancer subtypes, focusing on anastrozole and tamoxifen impacts. The present methods inadequately monitor and forecast patient reactions to these treatments, leaving individuals vulnerable to the potential adverse effects of these medications. This research investigated glycan structural changes by following patients for up to 9 months. The protocol involved a series of automated steps including IgG isolation, protein denaturation, glycan labelling, purification, and final analysis using capillary gel electrophoresis with laser-induced fluorescence. The results suggested the significant role of glycan modifications in breast cancer progression, revealing distinctive trends in how anastrozole and tamoxifen elicit varied responses. The findings indicate anastrozole's association with reduced sialylation and increased core fucosylation, while tamoxifen correlated with increased sialylation and decreased core fucosylation. These observations suggest potential immunomodulatory effects: anastrozole possibly reducing inflammation and tamoxifen impacting immune-mediated cytotoxicity. This study strongly emphasizes the importance of considering specific glycan traits to comprehend the dynamic mechanisms driving breast cancer progression and the effects of targeted therapies. The nuanced differences observed in glycan modifications between these two treatments underscore the necessity for further comprehensive research aimed at thoroughly evaluating the long-term implications and therapeutic efficacy for breast cancer patients.

In Situ Glycan Analysis and Editing in Living Systems.

Besides proteins and nucleic acids, carbohydrates are also ubiquitous building blocks of living systems. Approximately 70% of mammalian proteins are glycosylated. Glycans not only provide structural support for living systems but also act as crucial regulators of cellular functions. As a result, they are considered essential pieces of the life science puzzle. However, research on glycans has lagged far behind that on proteins and nucleic acids. The main reason is that glycans are not direct products of gene coding, and their synthesis is nontemplated. In addition, the diversity of monosaccharide species and their linkage patterns contribute to the complexity of the glycan structures, which is the molecular basis for their diverse functions. Research in glycobiology is extremely challenging, especially for the in situ elucidation of glycan structures and functions. There is an urgent need to develop highly specific glycan labeling tools and imaging methods and devise glycan editing strategies. This Perspective focuses on the challenges of in situ analysis of glycans in living systems at three spatial levels (i.e., cell, tissue, and in vivo) and highlights recent advances and directions in glycan labeling, imaging, and editing tools. We believe that examining the current development landscape and the existing bottlenecks can drive the evolution of in situ glycan analysis and intervention strategies and provide glycan-based insights for clinical diagnosis and therapeutics.

Droplet Microfluidics with Capillary Electrophoresis for Glycan Biomarker Analysis.

Several glycoproteins are validated biomarkers of various diseases such as cancer, cardiovascular diseases, chronic alcohol abuse, or congenital disorders of glycosylation (CDG). In particular, CDG represent a group of more than 150 inherited diseases with varied symptoms affecting multiple organs. The distribution of glycans from target glycoprotein(s) can be used to extract information to help the diagnosis and possibly differentiate subtypes of CDG. Indeed, depending on the glycans and the proteins to which they are attached, glycans can play a very broad range of roles in both physical and biological properties of glycoproteins. For glycans in general, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has become a staple. Analysis of glycans with CE-LIF requires several sample preparation steps, including release of glycans from the target glycoprotein, fluorescent labeling of glycans, and purification of labeled glycans. Here, we describe the protocol for glycan sample treatment in a microfluidic droplet system prior to CE-LIF of labeled glycans. The microfluidic droplet approach offers full automation, sample, and reagent volume reduction and elimination of contamination from external environment.

Fluorogenic cell surface glycan labelling with fluorescence molecular rotor dyes and nucleic acid stains.

We show that covalent labelling of sialic acids on live cell surfaces or mucin increases the fluorescence of the fluorescence molecular rotors (FMRs) CCVJ, Cy3 and thioazole orange, enabling wash-free imaging of cell surfaces. Dual labelling with an FMR and an environmentally insensitive dye allows detection of changes that occur, for example, when cross-linking is altered.

Longer fatty acid-protected GalNAz enables efficient labeling of proteins in living cells with minimized S-glyco modification.

Metabolic glycoengineering provides a powerful tool to label proteins with chemical tags for cell imaging and protein enrichment. The structures of per-O-acetylation on unnatural sugars facilitate membrane permeability and increase cellular uptake and are widely used for metabolic glycan labeling. However, unexpected S-glyco modification was discovered via a non-enzymatic reaction with protein cysteines, which was initially conducted with the hydrolysis of anomeric acetate by esterase. Herein, we synthesized a series of GalNAz derivatives that were protected with various lengths of short-chain fatty acid, including acetate, propionate, butyrate, valerate and pivalate, to detect differences in labeling efficiencies and occurrence of S-glyco modification. Our results demonstrate that all the GalNAz derivatives could effectively label proteins in HeLa cells, except the pivalate group. Of note, But4GalNAz exhibited excellent labeling abilities compared with Ac4GalNAz from the results for western blot, flow cytometry and confocal laser scanning microscopy. Moreover, the results for the S-glyco-modification assay by western blot and chemoproteomic analysis indicated that But4GalNAz generated negligible unexpected labeling signals compared to Ac4GalNAz. Our study uncovers the distinct labeling efficiency of different protected groups on unnatural sugars, which provides an alternative strategy to explore novel glycan probes.

Cell-Surface Glycan Labeling and Sensing

Cell-surface glycans are abundant and complex and play a critical role in maintaining protein stability, regulating cell behavior, and participating in cell communication. Obtaining structural information on glycans in situ is helpful to further understand the role of glycans in the physiological and pathological processes of cells and the regulatory mechanism. To achieve this, we can use recognition or labeling strategies to convert the presence of glycans on the cell surface into signals that can be detected. Currently, many different types of in situ sensing strategies for glycans have been developed. The spatial control of the conversion process can realize the restriction of glycan detection to specific proteins, and the introduction of signal amplification technology into the conversion process can improve the sensitivity of sensing. In this paper, the recent progress of glycan labeling methods and sensing technology is reviewed, and the future development direction is prospected.

Spotlight-author's view for "Metabolic glycan labeling immobilizes dendritic cell membrane and enhances antitumorefficacy of dendritic cell vaccine".

Spotlight-author's view for "Metabolic glycan labeling immobilizes dendritic cell membrane and enhances antitumorefficacy of dendritic cell vaccine".

Traceless Protein-Selective Glycan Labeling and Chemical Modification.

Executing glycan editing at a molecular level not only is pivotal for the elucidation of complicated mechanisms involved in glycan-relevant biological processes but also provides a promising solution to potentiate disease therapy. However, the precision control of glycan modification or glyco-editing on a selected glycoprotein is by far a grand challenge. Of note is to preserve the intact cellular glycan landscape, which is preserved after editing events are completed. We report herein a versatile, traceless glycan modification methodology for customizing the glycoforms of targeted proteins (subtypes), by orchestrating chemical- and photoregulation in a protein-selective glycoenzymatic system. This method relies on a three-module, ligand-photocleavable linker-glycoenzyme (L-P-G) conjugate. We demonstrated that RGD- or synthetic carbohydrate ligand-containing conjugates (RPG and SPG) would not activate until after the ligand-receptor interaction is accomplished (chemical regulation). RPG and SPG can both release the glycoenzyme upon photoillumination (photoregulation). The adjustable glycoenzyme activity, combined with ligand recognition selectivity, minimizes unnecessary glycan editing perturbation, and photolytic cleavage enables precise temporal control of editing events. An altered target protein turnover and dimerization were observed in our system, emphasizing the significance of preserving the native physiological niche of a particular protein when precise modification on the carbohydrate epitope occurs.

Metabolic glycan labeling immobilizes dendritic cell membrane and enhances antitumor efficacy of dendritic cell vaccine

Dendritic cell (DC) vaccine was among the first FDA-approved cancer immunotherapies, but has been limited by the modest cytotoxic T lymphocyte (CTL) response and therapeutic efficacy. Here we report a facile metabolic labeling approach that enables targeted modulation of adoptively transferred DCs for developing enhanced DC vaccines. We show that metabolic glycan labeling can reduce the membrane mobility of DCs, which activates DCs and improves the antigen presentation and subsequent T cell priming property of DCs. Metabolic glycan labeling itself can enhance the antitumor efficacy of DC vaccines. In addition, the cell-surface chemical tags (e.g., azido groups) introduced via metabolic glycan labeling also enable in vivo conjugation of cytokines onto adoptively transferred DCs, which further enhances CTL response and antitumor efficacy. Our DC labeling and targeting technology provides a strategy to improve the therapeutic efficacy of DC vaccines, with minimal interference upon the clinical manufacturing process.

Site-Specific and Multiple Fluorogenic Metabolic Glycan Labeling and Glycoproteomic Profiling in Live Cells.

Multicolor labeling for monitoring the intracellular localization of the same target type in the native environment using chemical fluorescent dyes is a challenging task. This approach requires both bioorthogonal and biocompatible ligations with an excellent fluorescence signal-to-noise ratio. Here, we present a metabolic glycan labeling technique that uses homemade fluorogenic dyes to investigate glycosylation patterns in live cells. These dyes allowed us to demonstrate rapid and efficient simultaneous multilabeling of glycoconjugates with minimum fluorescence noise. Our results demonstrate that this approach is capable of not only probing sialylation and GlcNAcylation in cells but also specifically labeling the cell-surface and intracellular sialylated glycoconjugates in live cells. In particular, we performed site-specific dual-channel fluorescence imaging of extra and intracellular sialylated glycans in HeLa and PC9 cancer cells as well as identified fluorescently labeled sialylated glycoproteins and glycans by a direct enrichment approach combined with an MS-based proteomic analysis in the same experiment. In conclusion, this study provides multilabeling tools in cellular systems for simultaneous site-specific glycan imaging and glycoproteomic analysis to study potential cancer- and disease-associated glycoconjugates.

Abstract P1166: O-GlcNAcylation Regulates Protein Synthesis In Cardiomyocytes Through Effects On The Translational Regulator Eif2α

The regulated and reversible modification of Ser/Thr residues by O-linked N-Acetylglucosamine (O-GlcNAc) is termed O-GlcNAcylation. O-GlcNAc cycling on and off proteins is regulated by two conserved enzymes, the ‘writer’ O-GlcNAc Transferase (OGT) and the ‘eraser’ O-GlcNAcase (OGA). Crucially, O-GlcNAcylation is implicated in nutrient sensing, cell growth and stress adaptation. Despite these significant roles, the precise pathways impacted by O-GlcNAcylation and what key substrate proteins are involved remain incompletely understood. A hallmark of cardiomyocyte hypertrophy is the rapid production of new proteins that assemble into the contractile machinery of the growing myocyte. eIF2α is a rate-limiting factor in translation initiation. Interestingly, phosphorylation of eIF2α causes the arrest of general mRNA translation and favors the translation of stress-adaptive mRNAs. In the present work we investigated whether altering O-GlcNAcylation affects nascent protein synthesis (NPS) in hypertrophic cardiomyocytes. NPS was significantly elevated by the agonist phenylephrine, and this response was suppressed by OSMI-1 (OGT inhibitor). Moreover, we found that OGT-inhibited cells had increased phosphorylation of eIF2α. Additionally, we found increased protein levels of the stress adaptive factors Atf4 and Chop, whose mRNA translation is favored when eIF2α is phosphorylated. ISRIB, a small molecule designed to suppress the effects of phospho-eIF2α in altering translation, significantly prevented OSMI-1-induced accumulation of Atf4. Furthermore, we used metabolic labeling of glycans in cells, coupled with alkyne-azide ‘click’ chemistry and streptavidin pull-down, to show that eIF2α can be O-GlcNAcylated in cardiomyocytes. Furthermore, protein enrichment followed by mass spectrometry identified potential sites of O-GlcNAcylation on eIF2α. Taken together, these results highlight a novel role of O-GlcNAcylation on cardiomyocyte protein synthesis through the regulation of eIF2α. Future investigations will characterize the upstream mechanisms that promote eIF2α phosphorylation in OGT-inhibited cells and investigate the implications of direct eIF2α O-GlcNAcylation on hypertrophic protein synthesis in cardiomyocytes.