TEO-GalNAc: Tag-free Chemoenzymatic Enrichment and Quantitative Mapping of Tn/T Antigen Sialylation Occupancy via Ketone-Oxime Chemistry
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Mingliang Ye
PROVIDER: PXD075239 | iProX | Wed Mar 04 00:00:00 GMT 2026
REPOSITORIES: iProX
Similar Datasets
Project description:Chemoenzymatic labeling has greatly advanced O-GlcNAc proteomics, yet existing strategies using azide-modified donors (e.g., UDP-GalNAz) require multi-step click-based workflows that introduce substantial sample loss and limit sensitivity toward low-abundance targets. While recent one-step labeling with biotinylated UDP-GalNAc donors streamlines workflows, the irreversible avidin–biotin interaction prevents glycopeptide recovery and restricts site-specific analysis. To address these challenges, we developed a ketone-reversible O-GlcNAc enrichment (KRO-GlcNAc) strategy employing a ketone-functionalized UDP-GalNAc analog (UDP-GalNLev) for one-step enzymatic tagging. GalT1(Y289L) transfers the GalNLev moiety with >96% efficiency, eliminating intermediate reactions and minimizing handling-induced loss. Crucially, reversible oxime-based capture on hydroxylamine-functionalized beads followed by mild methoxyamine release not only overcomes the irreversible nature of reported ketone-hydroxylamine-biotin enrichment, but fundamentally address the broader limitations that originally necessitated the transition from ketone-based labeling to azide chemistry. Application of KRO-GlcNAc to HeLa cells identified 4,129 potential O-GlcNAcylation sites across 740 proteins, with ~30% being novel. Functional studies revealed that O-GlcNAcylation regulates nuclear-cytoplasmic shuttling of chromatin remodeler EP400, linking this modification to stress granule assembly. This platform thus provides a powerful and versatile tool for advancing O-GlcNAcylation research.
2026-01-06 | PXD072723 |
Project description:Carbohydrate Response Element-Binding Protein (ChREBP) is a transcription factor that activates key genes involved in glucose, fructose, and lipid metabolism in response to carbohydrate feeding, but its other potential roles in metabolic homeostasis have not been as well studied. We used liver-selective GalNAc-siRNA technology to suppress expression of ChREBP in rats fed a high fat/high sucrose diet and characterized hepatic and systemic responses by integrating transcriptomic and metabolomic analyses. GalNAc-siChREBP-treated rats had lower levels of multiple short-chain acyl CoA metabolites compared to rats treated with GalNAc-siCtrl containing a non-targeting siRNA sequence. These changes were related to a sharp decrease in free CoA levels in GalNAc-siChREBP treated-rats, accompanied by lower expression of transcripts encoding enzymes and transporters involved in CoA biosynthesis. These activities of ChREBP likely contribute to its complex effects on hepatic lipid and energy metabolism. While core enzymes of fatty acid (FA) oxidation are induced by ChREBP knockdown, accumulation of liver acylcarnitines and circulating ketones indicate diversion of acetyl CoA to ketone production rather than complete oxidation in the TCA cycle. Despite strong suppression of pyruvate kinase and activation of pyruvate dehydrogenase, pyruvate levels were maintained, likely via increased expression of pyruvate transporters, and decreased expression of lactate dehydrogenase and alanine transaminase. GalNAc-siChREBP treatment increased hepatic citrate and isocitrate levels while decreasing levels of distal TCA cycle intermediates. The drop in free CoA levels, needed for the 2-ketoglutarate dehydrogenase reaction, as well as a decrease in transcripts encoding the anaplerotic enzymes pyruvate carboxylase, glutamate dehydrogenase, and aspartate transaminase likely contributed to these effects. GalNAc-siChREBP treatment caused striking increases in PRPP and ZMP/AICAR levels, and decreases in GMP, IMP, AMP, NaNM, NAD(P), and NAD(P)H levels, accompanied by reduced expression of enzymes that catalyze late steps in purine and NAD synthesis. ChREBP suppression also increased expression of a set of plasma membrane amino acid transporters, possibly as an attempt to replenish TCA cycle intermediates. In sum, combining transcriptomic and metabolomic analyses has revealed regulatory functions of ChREBP that go well beyond its canonical roles in control of carbohydrate and lipid metabolism to now include mitochondrial metabolism and cellular energy balance.
2025-11-06 | GSE282614 | GEO
Project description:Tn antigen (Tn), a single N-acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser and Thr residues, is found on most solid tumors yet rarely detected in adult tissues: featuring it one of the most distinctive signatures of cancers. Although it is prevalent in cancers, Tn-glycosylation sites are not entirely clear owing to the lack of suitable technology. Knowing the Tn-glycosylation sites will spur the development of the new vaccines, diagnostics, and therapeutics of cancers. Here, we report a novel technology named EXoO-Tn for large-scale mapping of Tn-glycosylation sites. EXoO-Tn utilizes glycosyltransferase C1GalT1 and isotopically-labeled UDP-Gal(13C6) to tag and convert Tn to Gal(13C6)-Tn. This exquisite Gal(13C6)-Tn structure is recognized by a human-gut-bacterial enzyme, called OpeRATOR, that specifically cleaves N-termini of the Gal(13C6)-Tn-occupied Ser and Thr residues to yield site-containing glycopeptides. The enzymes C1GalT1 and OpeRATOR could be used concurrently in one-pot. The effectiveness of EXoO-Tn was evaluated by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. Bioinformatic analysis of the identified site-specific Tn-glycoproteins revealed conserved motif, cellular localization, relative position in proteins, and mapped site-specific Tn-glycoproteome in different studies. Given the significance of Tn in cancers, EXoO-Tn is anticipated to have broad utilities in clinical study of cancers.
2022-02-15 | PXD014390 | Pride
Project description:This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Kevin White mailto:kpwhite@uchicago.edu (Principal Investigator), Subhradip Karmakar mailto:subhradip@uchicago.edu (Project Lead), Nick Bild mailto:nbild@bsd.uchicago.edu (Data Analyst), Alina Choudhury mailto:achoudhury@uchicago.edu (Laboratory Technician), Marc Domanus mailto:mdomanus@anl.gov (Sequencing Technician at Argonne National Lab)). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu). This ENCODE track maps human transcription factor binding sites, genome-wide using second generation massively parallel sequencing. This mapping uses expressed transcription factors as GFP tagged fusion proteins after BAC (Bacterial artificial chromosomes) recombineering. The U. of Chicago and Max Planck Institute (Dresden) pipeline generates recombineered (recombination-mediated genetic engineering) BACs for the production of cell lines or animals that express fusion proteins from epitope tagged transgenes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols. (http://hgwdev/ENCODE/protocols/cell) Recombineering strategy: To facilitate high-throughput production of the transgenic constructs, the program BACFinder (Crowe, Rana et al. 2002) automatically selects the most suitable BAC clone for any given human gene and generates the sets of PCR primers required for tagging and verification (Poser, Sarov et al. 2008). Recombineering is used for tagging cassettes at either the N or C terminus of the protein. The N-terminal cassette has a dual eukaryotic-prokaryotic promoter (PGK-gb2) driving a neomycin-kanamycin resistance gene within an artificial intron inside the tag coding sequence. The selection cassette is flanked by two loxP sites and can be permanently removed by Cre recombinase-mediated excision. The C-terminal cassette contains the sequence encoding the tag followed by an internal ribosome entry site (IRES) in front of the neomycin resistance gene. In addition, a short bacterial promoter (Gb3) drives the expression of the neomycin-kanamycin resistance gene in E. coli. The tagging cassettes, containing 50 nucleotides of PCR-introduced homology arms are inserted into the BAC by recombineering, either behind the start codon (for the N-terminal tag) or in front of the stop codon (for the C-terminal tag) of the gene. E. coli cells that have successfully recombined the cassette are selected for kanamycin resistance in liquid culture. Each saturated culture from a specific recombineering reaction derived 10-200 independent recombination events. Checking two independent clones for each PCR through the tag insertion point, 97% (85/88) yielded a PCR product of the expected size. Most of the clones that failed to grow were missing the targeted genomic region. An estimated 10% of the BACs used are chimeric, rearranged or wrongly mapped. Thus, initial results indicate that the necessary recombineering steps can be carried out with high fidelity. The White lab produced all epitope tagged transcription and chromatin factor BACs, as well as the genome wide ChIP data and analysis. An application of this approach to the analysis of closely related paralogs (RARa and RARg) yielded transcription factors, chromatin factors, cell lines, ChIP chip data and ChIP-seq data (Hua, Kittler et al. Cell 2009). Such paralogous transcription factors often can not otherwise be distinguished by antibodies. Sample Preparation: ChIP DNA from samples are sheared to ~800bp using a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of 150-200bp are isolated by gel extraction and amplified using limited cycles of PCR. Sequencing System: Illumina GAIIx and HySeq next-generation sequencing produced all ChIP-seq data. Processing and Analysis Software: Raw sequencing reads are aligned using Bowtie version 0.12.5 (Langmead et al. 2009). The "-m 1" parameter is applied to suppress alignments mapping more than once in the genome. Reads are aligned to the UCSC hg19 assembly. Wiggle format signal files are generated with SPP 2.7.1 for R 2.7.1. Macs 1.3.7 is used to call peaks. The Macs parameters used vary by experiment. The White lab used goat anti-GFP antibody to perform ChIP in untagged K562 cells as a background control. The test IP was performed in the same way as the background control. Results are expressed as values of the test normalized to the background
2011-08-12 | E-GEOD-31363 | biostudies-arrayexpress
Project description:Tumors are prone to immune escape by developing strategies to evade immunity. ST6GalNAc-I is an O-glycosyltransferase, which catalyzes the addition of sialic acid onto the first sugar GalNAc (Tn) and results in the formation of Neu5Acα2, 6GalNAc (STn) carbohydrate antigen. SialylTn plays an important role in tumor progression, angiogenesis, and metastasis. RNA sequence analysis was performed using genetically engineered LUAD mouse model tumors (KrasG12D/+; Trp53R172H/+; Ad-Cre (KPA) and KrasG12D/+; Ad-Cre (KA)). We have performed RNA sequence analysis using KPA tumors (N=3), KA tumors (N=3), and littermate control lung tissues (N=3). In comparison to normal lung tissues, several genes were significantly overexpressed in KPA and KA-derived LUAD tumors, including Meg3, Slc7a5, Cxcr1, Awat1, Kng2, Gjb3, Mfi2, and Dlk1. More specifically, St6galnac-I was significantly overexpressed in aggressive-type KPA tumor tissues compared to KA and normal lung tissues. Expression of ST6GalNAc-I is associated with tumor progression and angiogenesis by immunosuppression. Mechanistically, ST6GalNAc-I promotes tumor cell sialylation that impacts tumor angiogenesis by altering mucin MUC5AC. Similarly, ST6GalNAc-I regulates Nectin2 sialylation which leads to immunosuppression and tumor progression. Overall, our study defined that ST6GalNAc-I is a critical molecule for tumor cell sialylation for disease aggressiveness.
2025-04-15 | GSE290957 | GEO
Project description:S-fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human disease. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterization of endogenously attached fatty acids is still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods.
2018-03-28 | PXD008399 | Pride
Project description:Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&Tag, a target antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) recognized by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&Tag eliminates dependence on traditional target antibodies, achieving robust specificity and sensitivity with as few as 500 cells. It provides high-quality chromatin profiles, with improved signal-to-noise ratios and library quality compared with conventional antibody-based counterparts, while also enabling single-cell resolution (scAf-CUT&Tag). Applying Af-CUT&Tag to Hippo effectors (YAP1/TAZ) during liver regeneration revealed dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., Lpin1, Fasn) and heme clearance (Hpx, Trf). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&Tag in cell lines, bulk tissues, and single nuclei establishes it as a powerful tool for studying gene regulation in development, disease, and regeneration. Keywords: Antibody-Free CUT&Tag; Chromatin Binding; Epigenetic Profiling; Peptide-binder; Single-cell analysis
2024-12-15 | GSE283705 | GEO
Project description:Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&Tag, a target antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) recognized by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&Tag eliminates dependence on traditional target antibodies, achieving robust specificity and sensitivity with as few as 500 cells. It provides high-quality chromatin profiles, with improved signal-to-noise ratios and library quality compared with conventional antibody-based counterparts, while also enabling single-cell resolution (scAf-CUT&Tag). Applying Af-CUT&Tag to Hippo effectors (YAP1/TAZ) during liver regeneration revealed dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., Lpin1, Fasn) and heme clearance (Hpx, Trf). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&Tag in cell lines, bulk tissues, and single nuclei establishes it as a powerful tool for studying gene regulation in development, disease, and regeneration. Keywords: Antibody-Free CUT&Tag; Chromatin Binding; Epigenetic Profiling; Peptide-binder; Single-cell analysis
2024-12-15 | GSE283704 | GEO
Project description:Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&Tag, a target antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) recognized by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&Tag eliminates dependence on traditional target antibodies, achieving robust specificity and sensitivity with as few as 500 cells. It provides high-quality chromatin profiles, with improved signal-to-noise ratios and library quality compared with conventional antibody-based counterparts, while also enabling single-cell resolution (scAf-CUT&Tag). Applying Af-CUT&Tag to Hippo effectors (YAP1/TAZ) during liver regeneration revealed dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., Lpin1, Fasn) and heme clearance (Hpx, Trf). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&Tag in cell lines, bulk tissues, and single nuclei establishes it as a powerful tool for studying gene regulation in development, disease, and regeneration. Keywords: Antibody-Free CUT&Tag; Chromatin Binding; Epigenetic Profiling; Peptide-binder; Single-cell analysis
2024-12-15 | GSE283706 | GEO
Project description:We report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway (mut-AGX1/NahK/Nh-GalNAc-T2) that transforms an alkyne tagged sugar into the corresponding nucleotide-sugar. Only transfected cells incorporate the bioorthogonal sugar into glycoproteins in the presence of non-transfected cells. The incorporation of alkyne-sugar in glycoproteins allows the installation, by copper(I)-catalyzed reaction, of a biotinylated tag. Glycoproteins are enriched using Streptavidin, and on-bead digestion yielded a peptide fraction that is analysed by mass spectrometry.
2022-09-10 | PXD035438 | Pride