Diverse Post-translational Modifications Research Articles

Beyond glucose: The crucial role of redox signaling in β-cell metabolic adaptation

ObjectiveRedox signaling mediated by reversible oxidative cysteine thiol modifications is crucial for driving cellular adaptation to dynamic environmental changes, maintaining homeostasis, and ensuring proper function. This is particularly critical in pancreatic β-cells, which are highly metabolically active and play a specialized role in whole organism glucose homeostasis. Glucose stimulation in β-cells triggers signals leading to insulin secretion, including changes in ATP/ADP ratio and intracellular calcium levels. Additionally, lipid metabolism and reactive oxygen species (ROS) signaling are essential for β-cell function and health. MethodsWe employed IodoTMT isobaric labeling combined with tandem mass spectrometry to elucidate redox signaling pathways in pancreatic β-cells. ResultsGlucose stimulation significantly increases ROS levels in β-cells, leading to targeted reversible oxidation of proteins involved in key metabolic pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, pyruvate metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum (ER), and insulin secretion. Furthermore, the glucose-induced increase in reversible cysteine oxidation correlates with the presence of other post-translational modifications, including acetylation and phosphorylation. ConclusionsProper functioning of pancreatic β-cell metabolism relies on fine-tuned regulation, achieved through a sophisticated system of diverse post-translational modifications that modulate protein functions. Our findings demonstrate that glucose induces the production of ROS in pancreatic β-cells, leading to targeted reversible oxidative modifications of proteins. Furthermore, protein activity is modulated by acetylation and phosphorylation, highlighting the complexity of the regulatory mechanisms in β-cell function.

Integrative Multi-PTM Proteomics Reveals Dynamic Global, Redox, Phosphorylation, and Acetylation Regulation in Cytokine-treated Pancreatic Beta Cells.

Studying regulation of protein function at a systems level necessitates an understanding of the interplay among diverse post-translational modifications (PTMs). A variety of proteomics sample processing workflows are currently used to study specific PTMs but rarely characterize multiple types of PTMs from the same sample inputs. Method incompatibilities and laborious sample preparation steps complicate large-scale physiological investigations and can lead to variations in results. The single-pot, solid-phase-enhanced sample preparation (SP3) method for sample cleanup is compatible with different lysis buffers and amenable to automation, making it attractive for high-throughput multi-PTM profiling. Herein, we describe an integrative SP3 workflow for multiplexed quantification of protein abundance, cysteine thiol oxidation, phosphorylation, and acetylation. The broad applicability of this approach is demonstrated using cell and tissue samples, and its utility for studying interacting regulatory networks is highlighted in a time-course experiment of cytokine-treated β-cells. We observed a swift response in global regulation of protein abundances consistent with rapid activation of JAK-STAT and NF-κB signaling pathways. Regulators of these pathways as well as proteins involved in their target processes displayed multi-PTM dynamics indicative of a complex cellular response stages: acute, adaptation, and chronic (prolonged stress). PARP14, a negative regulator of JAK-STAT, had multiple co-localized PTMs that may be involved in intraprotein regulatory crosstalk. Our workflow provides a high-throughput platform that can profile multi-PTMomes from the same sample set, which is valuable in unraveling the functional roles of PTMs and their co-regulation.

Post-translational toxin modification by lactate controls Staphylococcus aureus virulence.

Diverse post-translational modifications have been shown to play important roles in regulating protein function in eukaryotes. By contrast, the roles of post-translational modifications in bacteria are not so well understood, particularly as they relate to pathogenesis. Here, we demonstrate post-translational protein modification by covalent addition of lactate to lysine residues (lactylation) in the human pathogen Staphylococcus aureus. Lactylation is dependent on lactate concentration and specifically affects alpha-toxin, in which a single lactylated lysine is required for full activity and virulence in infection models. Given that lactate levels typically increase during infection, our results suggest that the pathogen can use protein lactylation as a mechanism to increase toxin-mediated virulence during infection.

Untangling the Complexity and Impact of Tau Protein Ubiquitination.

The microtubule-associated protein tau is an intrinsically disordered protein highly expressed in neuronal axons. In healthy neurons, tau regulates microtubule dynamics and neurite outgrowth. However, pathological conditions can trigger aberrant tau aggregation into insoluble filaments, a hallmark of neurodegenerative disorders known as tauopathies. Tau undergoes diverse posttranslational modifications (PTMs), suggesting complex regulation and potentially varied functions. Among PTMs, the role and mechanisms of ubiquitination in physiology and disease have remained enigmatic. The past three decades have witnessed the emergence of key studies on tau protein ubiquitination. In this concept, we discuss how these investigations have begun to shed light on the ubiquitination patterns of physiological and pathological tau, the responsible enzymatic machinery, and the influence of ubiquitination on tau aggregation. We also provide an overview of the semi-synthetic methods that have enabled in vitro investigations of conformational transitions of tau induced by ubiquitin modification. Finally, we discuss future perspectives in the field necessary to elucidate the molecular mechanisms of tau ubiquitination and clearance.

7340 Elucidating the Role of Steroid Hormone Receptors in Anaplastic Thyroid Carcinoma and Pancreatic Ductal Carcinoma Models

Abstract Disclosure: O.M. Stockert: None. C.A. Lange: None. Steroid hormones play important roles in modulating transcription mechanisms in a variety of cancers, including breast cancer, prostate cancer, and ovarian cancer. These interactions are highly context-dependent and regulated by diverse ligands, cofactors, and dynamic post-translational modifications that occur in response to the activation of stress-sensing and oncogenic signaling pathways. Numerous other cancers are associated with elevated steroid hormones, steroid hormone receptors, or both, but have not been studied to explore the possibility of leveraging steroid or steroid receptor (SR)-modulated transcriptional regulation to combat cancerous growth. Two of these cancers are pancreatic ductal adenocarcinoma (PDAC) and anaplastic thyroid carcinoma (ATC). PDAC is an aggressive cancer with a very poor 5-year survival of 8-11%. It is also rapidly growing in incidence and has been shown to be associated with other increasing health conditions, including diabetes and obesity. PDAC is associated with metabolic reprogramming and elevated Progesterone Receptor (PR), Glucocorticoid Receptor (GR), and Mineralocorticoid Receptor (MR) expression. ATC is another aggressive cancer with less than 10% 6-month survival from diagnosis. Like PDAC, ATC has a rapidly increasing incidence and is associated with obesity and high-fat diets. ATC is also associated with elevated cholesterol biosynthesis and elevated GR expression. Herein, we probed public data sets and screened a panel of PDAC and ATC models to demonstrate the expression of mixed SRs. In this study, we take an initial exploration of the hypothesis that related SRs (such as PR, GR, and/or MR) contribute to PDAC and ATC progression and that post-translational modifications, ligand binding, and/or selected cofactors modulate SR actions in the context of activated signaling pathways associated with these aggressive cancers. This study gives us a preliminary understanding of the potential role of steroid hormones and their receptors in the aggressive biology of two deadly and increasingly prevalent endocrine cancers in aging and obese populations. Presentation: 6/3/2024

Activity-Based Acylome Profiling with N-(Cyanomethyl)-N-(phenylsulfonyl)amides for Targeted Lysine Acylation and Post-Translational Control of Protein Function in Cells.

Lysine acylations are ubiquitous and structurally diverse post-translational modifications that vastly expand the functional heterogeneity of the human proteome. Hence, the targeted acylation of lysine residues has emerged as a strategic approach to exert biomimetic control over the protein function. However, existing strategies for targeted lysine acylation in cells often rely on genetic intervention, recruitment of endogenous acylation machinery, or nonspecific acylating agents and lack methods to quantify the magnitude of specific acylations on a global level. In this study, we develop activity-based acylome profiling (ABAP), a chemoproteomic strategy that exploits elaborate N-(cyanomethyl)-N-(phenylsulfonyl)amides and lysine-centric probes for site-specific introduction and proteome-wide mapping of posttranslational lysine acylations in human cells. Harnessing this framework, we quantify various artificial acylations and rediscover numerous endogenous lysine acylations. We validate site-specific acetylation of target lysines and establish a structure-activity relationship for N-(cyanomethyl)-N-(phenylsulfonyl)amides in proteins from diverse structural and functional classes. We identify paralog-selective chemical probes that acetylate conserved lysines within interferon-stimulated antiviral RNA-binding proteins, generating de novo proteoforms with obstructed RNA interactions. We further demonstrate that targeted acetylation of a key enzyme in retinoid metabolism engenders a proteoform with a conformational change in the protein structure, leading to a gain-of-function phenotype and reduced drug potency. These findings underscore the versatility of our strategy in biomimetic control over protein function through targeted delivery and global profiling of endogenous and artificial lysine acylations, potentially advancing therapeutic modalities and our understanding of biological processes orchestrated by these post-translational modifications.

Mass Spectral Feature Analysis of Ubiquitylated Peptides Provides Insights into Probing the Dark Ubiquitylome.

Ubiquitylation is a structurally and functionally diverse post-translational modification that involves the covalent attachment of the small protein ubiquitin to other protein substrates. Trypsin-based proteomics is the most common approach for globally identifying ubiquitylation sites. However, we estimate that such methods are unable to detect ∼40% of ubiquitylation sites in the human proteome, i.e., "the dark ubiquitylome", including many important for human health and disease. In this meta-analysis of three large ubiquitylomic data sets, we performed a series of bioinformatic analyses to assess experimental features that could aid in uniquely identifying site-specific ubiquitylation events. Spectral predictions from Prosit were compared to experimental spectra of tryptic ubiquitylated peptides, revealing previously uncharacterized fragmentation of the diGly scar. Analysis of the LysC-derived ubiquitylated peptides reveals systematic, multidimensional peptide fragmentation, including diagnostic b-ions from fragmentation of the LysC ubiquitin scar. Comprehensively, these findings provide diagnostic spectral signatures of modification events that could be applied to new analysis methods for nontryptic ubiquitylomics.

Comprehensive posttranslational modifications in the testis-specific histone variant H3t protein validated in tagged knock-in mice

During the development of multicellular organisms and cell differentiation, the chromatin structure in the cell nucleus undergoes extensive changes, and the nucleosome structure is formed by a combination of various histone variants. Histone variants with diverse posttranslational modifications are known to play crucial roles in different regulatory functions. We have previously reported that H3t, a testis-specific histone variant, is essential for spermatogenesis. To elucidate the function of this chromatin molecule in vivo, we generated knock-in mice with a FLAG tag attached to the carboxyl terminus of H3t. In the present study, we evaluated the utility of the generated knock-in mice and comprehensively analyzed posttranslational modifications of canonical H3 and H3t using mass spectrometry. Herein, we found that H3t-FLAG was incorporated into spermatogonia and meiotic cells in the testes, as evidenced by immunostaining of testicular tissue. According to the mass spectrometry analysis, the overall pattern of H3t-FLAG posttranslational modification was comparable to that of the control H3, while the relative abundances of certain specific modifications differed between H3t-FLAG and the control bulk H3. The generated knock-in mice could be valuable for analyzing the function of histone variants in vivo.

Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry

The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuclear proteomic-states. The data demonstrate the heterogeneity of cell-states and signaling dependencies at the single-cell level and reveal epigenetic drug-induced changes in single nuclei. This approach enables the exploration of previously uncharted single-cell and organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.

Tubulin code eraser CCP5 binds branch glutamates by substrate deformation.

Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes1. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification2. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family1. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function3-9, and mutations in CCPs lead to human disease10-13. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant β-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.

Protein complex heterogeneity and topology revealed by electron capture charge reduction and surface induced dissociation.

We illustrate the utility of native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), for the characterization of protein complex topology and glycoprotein heterogeneity. ECCR efficiently reduces the charge states of tetradecameric GroEL, illustrating Orbitrap m/z measurements to greater than 100,000 m/z. For pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) reduces the charge states and yields more topologically informative fragmentation. This is the first demonstration that ECCR yields more native-like SID fragmentation. ECCR also significantly improved mass and glycan heterogeneity measurements of heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer. Protein glycosylation is important for structural and functional properties and plays essential roles in many biological processes. The immense heterogeneity in glycosylation sites and glycan structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Without ECCR, average mass determination of glycoprotein complexes is available only through charge detection mass spectrometry or mass photometry. With narrow m/z selection windows followed by ECCR, multiple glycoform m/z values are apparent, providing quick global glycoform profiling and providing a future path for glycan localization on individual intact glycoforms.

Glycoproteomics: Charting new territory in mass spectrometry and glycobiology.

Glycosylation is an incredibly common and diverse post-translational modification that contributes widely to cellular health and disease. Mass spectrometry is the premier technique to study glycoproteins; however, glycoproteomics has lagged behind traditional proteomics due to the challenges associated with studying glycosylation. For instance, glycans dissociate by collision-based fragmentation, thus necessitating electron-based fragmentation for site-localization. The vast glycan heterogeneity leads to lower overall abundance of each glycopeptide, and often, ion suppression is observed. One of the biggest issues facing glycoproteomics is the lack of reliable software for analysis, which necessitates manual validation and serves as a massive bottleneck in data processing. Here, I will discuss each of these challenges and some ways in which the field is attempting to address them, along with perspectives on how I believe we should move forward.

Dynein Light Intermediate Chains Exhibit Different Arginine Methylation Patterns.

The motor protein dynein is integral to retrograde transport along microtubules and interacts with numerous cargoes through the recruitment of cargo-specific adaptor proteins. This interaction is mediated by dynein light intermediate chain subunits LIC1 (DYNC1LI1) and LIC2 (DYNC1LI2), which govern the adaptor binding and are present in distinct dynein complexes with overlapping and unique functions. Using bioinformatics, we analyzed the C-terminal domains (CTDs) of LIC1 and LIC2, revealing similar structural features but diverse post-translational modifications (PTMs). The methylation status of LIC2 and the proteins involved in this modification were examined through immunoprecipitation and immunoblotting analyses. The specific methylation sites on LIC2 were identified through a site-directed mutagenesis analysis, contributing to a deeper understanding of the regulatory mechanisms of the dynein complex. We found that LIC2 is specifically methylated at the arginine 397 residue, a reaction that is catalyzed by protein arginine methyltransferase 1 (PRMT1). The distinct PTMs of the LIC subunits offer a versatile mechanism for dynein to transport diverse cargoes efficiently. Understanding how these PTMs influence the functions of LIC2, and how they differ from LIC1, is crucial for elucidating the role of dynein-related transport pathways in a range of diseases. The discovery of the arginine 397 methylation site on LIC2 enhances our insight into the regulatory PTMs of dynein functions.

Integration of Web-Based Tools to Visualize, Integrate, and Interpret Glycogene Expression and Glycomics Data.

Glycosylation is the most abundant and diverse post-translational modification occurring on proteins. Glycans play important roles in modulating cell adhesion, growth, development, and differentiation. Changes in glycosylation affect protein structure and function and contribute to disease processes. Therefore, understanding glycosylation patterns is key for the identification of targets for the diagnosis of diseases, cellular states, and therapy. Glycosylation is a nontemplate-driven process governed by the action of numerous enzymes and substrate availability that varies among cell types and species. Therefore, qualitative and quantitative assessment of global glycosylation and individual glycans remains challenging because it requires integration of multiple complex data types. Glycan structure and quantity data are often integrated with assessments of gene expression to aid contextualization of observed glycosylation changes within biological processes. However, correlating glycogene expression to the glycan structure is challenging because transcriptional changes may not always concur with the final gene product; there is often a lack of information on nucleotide sugar pools, and the final glycan structure is the result of many different glycogenes acting in concert. To overcome these challenges, interactive online tools are emerging as key resources for facilitating the analysis and integration of glycomics and glycogene expression data. Importantly, these tools work in concurrence with glycan biosynthetic schemes and therefore provide a clear indication of the molecular pathways where the glycan and glycogene are involved. In this chapter, we describe the applications of four freely available online tools that can be used for integrated visualization, interpretation, and presentation of RNAseq and glycomics results.

A Novel Gene Signature Associated with Protein Post-translational Modification to Predict Clinical Outcomes and Therapeutic Responses of Colorectal Cancer.

Accumulated evidence highlights the biological significance of diverse protein post-translational modifications (PTMs) in tumorigenicity and progression of colorectal cancer (CRC). In this study, ten PTM patterns (ubiquitination, methylation, phosphorylation, glycosylation, acetylation, SUMOylation, citrullination, neddylation, palmitoylation, and ADP-ribosylation) were analyzed for model construction. A post-translational modification index (PTMI) with a 14-gene signature was established. CRC patients with high PTMI had a worse prognosis after validating in nine independent datasets. By incorporating PTMI with clinical features, a nomogram with excellent predictive performance was constructed. Two molecular subtypes of CRC with obvious difference in survival time were identified by unsupervised clustering. Furthermore, PTMI was related to known immunoregulators and key tumor microenvironment components. Low-PTMI patients responded better to fluorouracil-based chemotherapy and immune checkpoint blockade therapy compared to high-PTMI patients, which was validated in multiple independent datasets. However, patients with high PTMI might be sensitive to bevacizumab. In short, we established a novel PTMI model by comprehensively analyzing diverse post-translational modification patterns, which can accurately predict clinical prognosis and treatment response of CRC patients.

Intrinsic Disorder in the Human Tear Proteome.

We aimed to characterize the proteome of human tears and assess for the presence of intrinsically disordered proteins (IDPs). IDPs, despite lacking a rigid three-dimensional structure, maintain biological functionality and could shed light on the molecular interactions within tears. We analyzed a dataset of 1475 proteins identified in the tear film of three healthy subjects. We employed several computational tools, including the Compositional Profiler, Rapid Intrinsic Disorder Analysis Online, Search Tool for the Retrieval of Interacting Genes, and Database of Disordered Protein Predictors to evaluate the intrinsic disorder, protein interactions, and functional characterization of the disordered regions within this proteome. Our analysis showed a notable inclination toward intrinsic disorder. Two out of 10 order-promoting residues and five out of 10 disorder-promoting residues were found enriched. Using the Predictor of Natural Disordered Regions (PONDR) VSL2 output, 95% of these proteins were classified as highly or moderately disordered. We revealed an extensive protein-protein interaction network with significant interaction enrichment. The most disordered proteins exhibited higher disorder binding sites and diverse posttranslational modifications compared to the most ordered ones. To the best of our knowledge, our study is the first comprehensive analysis of intrinsic disorder in the human tear film proteome, and it revealed an abundance of IDPs and their role in protein function and interaction networks. These findings suggest that variations in the intrinsic disorder of a tear film could be impacted by systemic and ocular conditions, offering promising avenues for disease biomarker identification and drug target development. Further research is needed to understand the implications of these findings in human health and disease.

RAGE Against the Glycation Machine in Synucleinopathies: Time to Explore New Questions.

Oligomerization and aggregation of misfolded forms of α-synuclein are believed to be key molecular mechanisms in Parkinson's disease (PD) and other synucleinopathies, so extensive research has attempted to understand these processes. Among diverse post-translational modifications that impact α-synuclein aggregation, glycation may take place at several lysine sites and modify α-synuclein oligomerization, toxicity, and clearance. The receptor for advanced glycation end products (RAGE) is considered a key regulator of chronic neuroinflammation through microglial activation in response to advanced glycation end-products, such as carboxy-ethyl-lysine, or carboxy-methyl-lysine. The presence of RAGE in the midbrain of PD patients has been reported in the last decades and this receptor was proposed to have a role in sustaining PD neuroinflammation. However, different PD animal models demonstrated that RAGE is preferentially expressed in neurons and astrocytes, while recent evidence demonstrated that fibrillar, non-glycated α-synuclein binds to RAGE. Here, we summarize the available data on α-synuclein glycation and RAGE in the context of PD, and discuss about the questions yet to be answered that may increase our understanding of the molecular bases of PD and synucleinopathies.

Oxidative Release of O-Glycans under Neutral Conditions for Analysis of Glycoconjugates Having Base-Sensitive Substituents.

Protein O-glycosylation is one of the most diverse post-translational modifications. A critical step in the analysis of O-glycomes is the release of glycans from glycoconjugates. Current release methods rely mainly on β-elimination, which can result in peeling reactions and loss of base-sensitive functionalities leading to misidentification of glycans. To address this challenge, well-defined synthetic glycopeptides were used to establish a robust workflow for the oxidative release of O-glycans suitable for glycomics. Treatment of O-glycopeptides with neutralized hypochlorite resulted in the selective formation of lactic/glycolic acid glycosides, thereby retaining unique information of the parent amino acid (serine/threonine) that is lost by β-elimination. It locks the glycan in a closed ring configuration, thereby preventing peeling, and furthermore, the carboxylate of the anomeric tag promotes ionization in negative ion mode mass spectrometry, thereby increasing signal intensities. Labile modifications such as sialic acids, sulfates, and acetyl esters are maintained during the release procedure. The promise of the approach was demonstrated by the analysis of O-glycans from bovine submaxillary mucin, which identified mono- and di-O-acetylated sialoglycans as well as previously undetected tri-O-acetylated and sulfated glycans. The use of well-defined glycopeptide standards made it also possible to identify reaction intermediates, which in turn allowed us to postulate a reaction mechanism for oxidative O-glycan release under neutral conditions.

Low-input chromatin profiling reveals inter- and intra-lineage differences across immune cell types

Abstract Transcriptional regulation in cells is governed by myriad traits that allow specific responses to ever-changing environments. Inclusive of these regulatory factors are the histone proteins that compact the genome while additionally being decorated with diverse post-translational modifications (PTM) suggested to direct the cellular machinery to express / repress lineage- and context-specific loci. Our consortium recently performed a detailed analysis of chromatin accessibility (ATACseq) across multiple mouse immunocyte populations, which advanced our understanding of how cis-regulatory elements (CRE) control transcriptional programs in rarer immune cell types. Understanding the role of histone PTMs in this process has remained elusive due to the high cell-input demands of the gold-standard assay: ChIPseq. To this end, we developed a streamlined CUT&RUN approach to support robust profiling at 10K cells per target in ~100 sorted mouse immunologic populations. We have used this to map histone PTM states characteristic of active (H3K4me1/3, H3K27ac, H3K36me3) or repressed (H3K27me3) chromatin and architectural factors associated with genome organization (H3.3, CTCF), in tandem with RNA- and ATAC-seq. We further dissected the effect of genomic variation by utilizing a B6xCast/EiJ F1 hybrid mouse to understand how CRE variation at each allele independently impacts transcriptional regulation. Taken together, our data provides a deeper understanding of transcriptional regulation in the mouse immune system across differentiation and perturbation states.

Multi-comparative Thermal Proteome Profiling Uncovers New O-GlcNAc Proteins in a System-wide Method

Among diverse protein post-translational modifications, O-GlcNAcylation, a simple but essential monosaccharide modification, plays crucial roles in cellular processes and is closely related to various diseases. Despite its ubiquity in cells, properties of low stoichiometry and reversibility are hard nuts to crack in system-wide research of O-GlcNAc. Herein, we developed a novel method employing multi-comparative thermal proteome profiling for O-GlcNAc transferase (OGT) substrate discovery. Melting curves of proteins under different treatments were profiled and compared with high reproducibility and consistency. Consequently, proteins with significantly shifted stabilities caused by OGT and uridine-5'-diphosphate N-acetylglucosamine were screened out from which new O-GlcNAcylated proteins were uncovered.