O-GlcNAc Glycosylation Proteomics
O-linked N-acetylglucosamine (O-GlcNAc) modification is a crucial post-translational modification (PTM) of proteins, wherein a single N-acetylglucosamine molecule is attached via a β-glycosidic bond to hydroxyl groups on serine (Ser) or threonine (Thr) residues of proteins. As the only glycosylation modification occurring inside cells, O-GlcNAc modification is widely present in the nucleus, cytoplasm, and mitochondria, participating in cellular signal transduction processes.
O-GlcNAc modification has the following characteristics:
- Widespread Distribution: Protein O-GlcNAc glycosylation exists in all metazoans, including animals, insects, and plants; this modification is also found in some bacteria, fungi, and viruses
- Dynamically Reversible: The addition and removal of O-GlcNAc are precisely regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) respectively, forming a highly reversible dynamic modification
- Multi-level Regulation: Operating in a species-, tissue/cell-, protein-, and site-specific manner, O-GlcNAc plays key regulatory roles in virtually all cellular processes including metabolism, transcriptional control, signal transduction, and protein stability
- Metabolic Sensing: Functioning as a cellular energy metabolism sensor, responding to the uptake and utilization of cellular glucose and amino acids
Aberrant O-GlcNAc modifications are closely associated with multiple important diseases, including cancer, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, etc.), metabolic diseases (diabetes, cardiovascular disease), and immune-related disorders. In-depth investigation of O-GlcNAc modifications is crucial for elucidating disease mechanisms and developing novel therapeutic strategies.
Service Offerings
We provide comprehensive O-GlcNAc glycosylation proteomics analysis services based on liquid chromatography-high-resolution mass spectrometry (LC-MS/MS) technology, with the following main content:
| Service Type | Service Description |
| O-GlcNAc Site Identification | Identify over 3,000 O-GlcNAc modification sites in a single sample, achieve amino acid-level precision in modification localization, discover previously unknown O-GlcNAc modification sites |
| O-GlcNAc Glycan Structure Analysis | Perform fine chemical structure identification of modified glycans, qualitatively analyze monosaccharide composition, quantitatively analyze relative/absolute glycan expression levels |
| Differential O-GlcNAc Modification Expression Analysis | Quantitatively compare O-GlcNAc modification abundance under different biological conditions, perform GO enrichment and pathway analysis of modified proteins, clustering and association analysis of modification expression patterns across samples |
| O-GlcNAc and Phosphorylation Dual Modification Analysis | Simultaneously analyze O-GlcNAc and phosphorylation modifications on individual proteins, reveal interactions and cross-regulation between the two modifications, deeply elucidate protein function regulation mechanisms |
| Protein Interaction and Functional Annotation | Construct interaction networks based on modified proteins, preliminary screening of disease-related protein biomarkers, annotation and biological significance interpretation of identified proteins |
Service Advantages
Our O-GlcNAc glycosylation proteomics analysis service offers distinct advantages. First, we employ chemical enzymatic labeling and bioorthogonal click chemistry enrichment strategies with high efficiency and strong selectivity, utilizing photolabile probes to achieve efficient release of labeled glycopeptides, effectively resolving the challenges of O-GlcNAc's low abundance and susceptibility to hydrolysis. Second, through high-resolution mass analyzers and multiple mass spectrometry fragmentation modes, we ensure experimental result reproducibility and stability while identifying numerous modification sites. Additionally, we support multiple quantitative methods including peak area-based labeled quantitation, label-free quantitation, and isotopic labeling quantitation, providing precise quantitation of both relative and absolute expression levels of modified proteins. Finally, we leverage tandem mass spectrometry data to infer O-GlcNAc glycan structures, distinguish among different types of glycan structural isomers, and provide structural-level support for glycosylation biology research.
Workflow
We extract proteins from input samples, determine protein concentration using the BCA method, and assess protein integrity and purity by SDS-PAGE gel imaging.
Proteins undergo denaturation, reduction, and alkylation treatment, followed by specific cleavage with trypsin or other alkaline proteases to decompose intact proteins into peptide mixtures.
After enzymatic digestion, peptide mixtures are subjected to chemical labeling, then O-GlcNAc-modified glycopeptides are enriched using hydrophilic interaction chromatography or solid-phase extraction, followed by photocleavage or enzymatic release.
Enriched samples are separated via high-performance liquid chromatography, analyzed using high-resolution mass spectrometry, and high-quality mass spectrometry data is obtained.
Raw mass spectrometry data is processed including peptide identification, protein inference, modification site confirmation and localization, database searching, and data quality control.
We perform characterization analysis of O-GlcNAc modification sites, calculate modification expression differences, conduct GO enrichment and pathway analysis, construct protein interaction networks, and provide in-depth data interpretation.
Application
O-GlcNAc glycosylation proteomics services are widely applied in signal transduction and gene expression regulation research, cellular metabolism studies, protein quality control, cancer research, neurodegenerative diseases, metabolic diseases, cardiovascular disease research, and other fields, encompassing diverse sample types including tissues from various animal models (mice, rats, Arabidopsis, etc.), human-derived cells and tissues, patient body fluid samples, primary cells and cell lines, as well as in vitro cell culture systems.
