diff --git a/README.md b/README.md index 485bf9d..b6581b5 100644 --- a/README.md +++ b/README.md @@ -1 +1,101 @@ -# OGlycoTM \ No newline at end of file +# Systematic Quantification of Protein *O*-GlcNAcylation under *N*-Glycosylation Inhibition + +[![Published in Analytical Chemistry](https://img.shields.io/badge/Anal.%20Chem.-2026%2C%2098%2C%2015689–15699-B31B1B)](https://doi.org/10.1021/acs.analchem.6c00972) +[![DOI](https://img.shields.io/badge/DOI-10.1021%2Facs.analchem.6c00972-blue)](https://doi.org/10.1021/acs.analchem.6c00972) +[![Data: PXD073249](https://img.shields.io/badge/ProteomeXchange-PXD073249-1f9c5a)](https://www.ebi.ac.uk/pride/archive/projects/PXD073249) +[![License: MIT](https://img.shields.io/badge/Code%20License-MIT-yellow)](LICENSE) +[![Open Access CC BY 4.0](https://img.shields.io/badge/Article-CC%20BY%204.0-lightgrey)](https://creativecommons.org/licenses/by/4.0/) + +Data-analysis code and figures for: + +> **Longping Fu, Kejun Yin, Xing Xu, and Ronghu Wu.** *Systematic Quantification of Protein O-GlcNAcylation Reveals Common and Cell-Type-Specific Responses to N-Glycosylation Inhibition in Human Cells.* **Analytical Chemistry** 2026, 98, 15689–15699. DOI: [10.1021/acs.analchem.6c00972](https://doi.org/10.1021/acs.analchem.6c00972) + +

+ Table of Contents graphic: metabolic labeling and multiplexed proteomics of O-GlcNAcylation in HEK293T, HepG2, and Jurkat cells under N-glycosylation inhibition, resolving common regulation, cell-type-specific responses, and site-specific structural analysis +

+ +## Overview + +Protein *O*-GlcNAcylation and *N*-glycosylation are two of the most abundant and functionally important glycosylation types in human cells, and both help regulate protein activity, stability, and signaling. *O*-GlcNAcylation — the reversible attachment of a single *N*-acetylglucosamine to serine, threonine, or tyrosine residues of nuclear and cytoplasmic proteins — is a well-established stress sensor, yet how it responds when *N*-glycosylation is perturbed had not been characterized comprehensively or at site-level resolution. Because the two modifications share a nucleotide-sugar precursor (UDP-GlcNAc) and *N*-glycosylation defects trigger ER stress, a crosstalk between them is expected but poorly mapped. + +Here, we combined metabolic labeling, bioorthogonal chemistry, and multiplexed (TMT) proteomics to comprehensively and site-specifically quantify protein *O*-GlcNAcylation in three human cell types — **HEK293T, HepG2, and Jurkat** — while inhibiting *N*-glycosylation with **tunicamycin**. Across the cells, more than 1,000 *O*-GlcNAcylated proteins (1,109 total) were identified and quantified. The results reveal both a **common** program shared across cell types and distinct **cell-type-specific** responses to *N*-glycosylation inhibition, and further show that the **local structural context of an *O*-GlcNAc site** is a key determinant of how much it changes. This repository contains the code used to process the mass spectrometry results and generate every figure and supporting table in the paper. + +## Highlights + +- **Dual-modification-resolved workflow.** Metabolic labeling with Ac₄GalNAz, click-chemistry enrichment, and a galactose-oxidase (GAO) step chemically separate *O*-GlcNAc (+528.2859 Da) from *O*-GalNAc / Tn antigen (+555.2968 Da), a 27 Da mass shift that lets the two structurally similar modifications be distinguished and quantified by MS. +- **1,109 *O*-GlcNAcylated proteins** identified and quantified across HEK293T, HepG2, and Jurkat cells, with 402 commonly detected in all three. +- **Common response to *N*-glycosylation inhibition.** Commonly up-regulated *O*-GlcNAcylated proteins are enriched for translation regulation, glucose response, and RNA splicing; commonly down-regulated ones are enriched for stress-granule assembly and stress-response regulation. +- **Cell-type-specific response.** In Jurkat cells, up-regulated *O*-GlcNAcylated proteins map to leukocyte proliferation, adhesion, and T-cell activation; in HEK293T cells they map to chaperone-mediated folding, cell-cycle, ribonucleotide metabolism, and ribosome biogenesis. +- **Site structure matters.** *O*-GlcNAcylation sites in structured protein regions undergo significantly larger abundance changes than sites in intrinsically disordered regions (IDRs), which stayed largely unaffected — suggesting structured-region sites are more responsive to cellular perturbation. + +## Method at a glance + +``` +Metabolic labeling & treatment HEK293T / HepG2 / Jurkat + tunicamycin (N-glyco inhibition) + Ac4GalNAz + │ +Click chemistry & enrichment CuAAC → Photocleavable-Biotin-Alkyne → NeutrAvidin capture → photocleavage (365 nm) + │ +TMT labeling & GAO oxidation TMT multiplexing; galactose oxidase distinguishes O-GlcNAc (+528.2859) vs O-GalNAc (+555.2968) + │ +LC-MS/MS High-pH HPLC (12 fractions) → Orbitrap Eclipse Tribrid, HCD-pd-EThcD + │ +Database search & analysis FragPipe + O-Pair Search → differential analysis (|log2(Tuni/Ctrl)| > 0.5, adj. P < 0.05) +``` + +## Repository structure + +``` +OGlycoTM/ +├── data_analysis/ R + Python analysis pipeline +│ ├── data_import.R Import search results +│ ├── data_normalization.R TMT normalization +│ ├── data_filtering.R Site / PSM filtering +│ ├── data_quantification.R O-GlcNAc quantification +│ ├── data_quantification_OGalNAc.R +│ ├── differential_analysis.R Tuni/Ctrl differential testing +│ ├── glycoprotein_classification.R +│ ├── Figure1.R … Figure6.R Main-text figure generation +│ ├── FigureS1.R, FigureS2.R Supporting figures +│ ├── Figure6E_*.py, Figure6F_*.py PyMOL structure panels (site structural context) +│ ├── generate_supporting_table_S1.R … S11 Supporting tables +│ ├── spectrum_annotator.py, Glycopeptide spectrum annotation +│ │ fragment_calculator.py +│ └── annotate_*.py, extract_*.py Spectrum extraction / annotation utilities +├── Manuscript/ Manuscript, supporting information, revisions +├── assets/ README figures (TOC graphic) +├── LICENSE MIT (code) +└── README.md +``` + +## Data availability + +The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the **PRIDE** partner repository under the dataset identifier **[PXD073249](https://www.ebi.ac.uk/pride/archive/projects/PXD073249)**. + +Per-cell-type identifications and abundance changes for *O*-GlcNAcylated proteins, *O*-GlcNAc sites, *O*-GalNAcylated proteins, and the whole proteome are provided as supporting-information tables with the published article. + +## Citation + +```bibtex +@article{Fu2026OGlcNAc, + title = {Systematic Quantification of Protein O-GlcNAcylation Reveals Common + and Cell-Type-Specific Responses to N-Glycosylation Inhibition in Human Cells}, + author = {Fu, Longping and Yin, Kejun and Xu, Xing and Wu, Ronghu}, + journal = {Analytical Chemistry}, + year = {2026}, + volume = {98}, + pages = {15689--15699}, + doi = {10.1021/acs.analchem.6c00972} +} +``` + +## Funding + +This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (**R35GM156318**). + +## License + +The code in this repository is released under the [MIT License](LICENSE). The associated article is open access under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). + +## Contact + +**Ronghu Wu** — School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA · [ronghu.wu@chemistry.gatech.edu](mailto:ronghu.wu@chemistry.gatech.edu) diff --git a/assets/table_of_contents.png b/assets/table_of_contents.png new file mode 100644 index 0000000..59c5a23 Binary files /dev/null and b/assets/table_of_contents.png differ