DIA-Based Glycoproteomics Services
- Sample Collection and Processing: Proteins are first extracted from biological samples, such as cells, tissues, or serum. During preparation, proteins are purified and concentrated to ensure high sensitivity during analysis.
- Glycoprotein Enrichment: Specific affinity capture methods (e.g., using lectin-based affinity reagents) are employed to enrich glycoproteins. This step isolates glycosylated proteins from complex samples.
- Liquid Chromatography (LC) Separation: High-performance liquid chromatography (HPLC) is used to separate peptides. After separation, the peptides are introduced into the mass spectrometer for analysis.
- Data-Independent Acquisition (DIA): In DIA mode, the mass spectrometer fragments all precursor ions within specified m/z windows instead of selecting specific ions. This method provides more comprehensive and consistent peptide detection, ideal for high-throughput analysis of complex samples.
- Peptide Analysis and Quantification: Data obtained through mass spectrometry are used to identify and quantify glycosylated peptides, revealing glycosylation sites and types of glycosylation.
- Data Processing: Specialized bioinformatics software analyzes the data, combining database searches to localize glycosylation sites. This includes peptide identification, glycosylation modification identification, and determining modification positions.
- Quantitative Analysis: The relative or absolute abundance of each peptide is calculated, allowing for the quantification of glycosylated proteins and revealing glycosylation changes under different conditions.
- Pathway Enrichment Analysis: Bioinformatics tools analyze the identified glycosylated proteins and map them to biological pathways, helping to uncover potential roles of glycosylation in various biological processes.
- Custom Reports: Detailed experimental data and analysis results are provided, including glycosylation sites, modification types, quantitative data, and pathway analysis.
Glycosylation is one of the most complex post-translational modifications in living organisms, directly regulating protein function, intercellular communication, and the mechanisms of diseases such as cancer, immune dysfunction, and neurodegenerative diseases. However, traditional glycoproteomics research is limited by the sensitivity, reproducibility, and quantitative capabilities of current technologies: data-dependent acquisition (DDA)-based methods often miss low-abundance glycopeptides, while conventional targeted approaches struggle to achieve high-throughput analysis. The advent of data-independent acquisition (DIA-MS) has brought a paradigm shift to glycoproteomics. By capturing all ion fragment information in an unbiased, full-scan mode and leveraging AI-driven glycopeptide library decoding capabilities, DIA-MS addresses the bottlenecks of analyzing glycosylation site heterogeneity (Micro-Heterogeneity) and glycan diversity (Macro-Heterogeneity).
Yue, Z. et al. Glycoconj J. 2023.
Figure 1. Alteration of Protein Glycosylation and Diseases
Today, MtoZ Biolabs, with its cutting-edge DIA-based platform, offers an industry-leading DIA-based glycoproteomics services that help you decode the molecular language of glycosylation with high precision and broad coverage. We provide a one-stop service from sample preparation to bioinformatics analysis, focusing on precise quantification and deep characterization of O/N-glycosylation, suitable for complex biological samples such as serum, tissues, and exosomes.
Analysis Workflow
1. Sample Preparation
2. Digestion
Digestion: The enriched glycoproteins are digested into smaller peptides using enzymes such as trypsin, facilitating mass spectrometry analysis.
3. DIA-MS Analysis
4. Data Analysis and Bioinformatics Processing
5. Result Interpretation and Reporting
Figure 2. The Workflow of DIA-Based Glycoproteomics
Through this workflow, our DIA-based glycoproteomics service offers comprehensive and sensitive analysis of glycosylation modifications in glycoprotein samples, shedding light on the potential roles of glycosylation in cellular processes.
Why Choose MtoZ Biolabs?
1. Outstanding Sensitivity and Data Integrity: DIA technology eliminates the "ion selection bias" of DDA, increasing the detection rate of low-abundance glycopeptides by more than 40%, especially suitable for low-concentration samples such as serum/plasma.
2. High-Throughput Quantification and Excellent Reproducibility: Cross-batch RSD <15%, supporting large-scale cohort studies (such as cancer early screening and drug efficacy monitoring).
3. Industry-Leading Glycan Analysis Depth: Capable of identifying complex modifications such as fucosylation and sialylation, and analyzing non-classical glycosylation sites.
4. Full-Chain Personalized Service: From experimental design to target validation (e.g., antibody development, CRISPR verification), we provide closed-loop support to accelerate the translation of results.
Case Study
1. Adapting Data-Independent Acquisition for Mass SpectrometryBased Protein Site-Specific N‐Glycosylation Analysis
This study explores the application of DIA in mass spectrometry-based protein site-specific N-glycosylation analysis. The DIA approach extracts glycopeptide-specific fragment ion chromatograms post-acquisition and aligns them with the precursor MS1 chromatograms, thereby overcoming the limitations of traditional DDA methods. DIA can efficiently identify low-abundance glycoforms in complex samples and supports the discovery of unexpected glycoform modifications. The study demonstrates that DIA outperforms DDA in both targeted and discovery modes, especially in cases where the sample is complex and the target glycoprotein is limited. DIA-based glycoproteomics services utilize advanced mass spectrometry techniques to comprehensively analyze site-specific protein glycosylation. It supports the identification of both targeted and unexpected glycoforms, even in complex biological samples.
Pan, KT. et al. Anal Chem. 2017.
Figure 3. DIA for N-glycopeptide Analysis
Applications
1. Disease Biomarker Discovery: Serum/tissue glycosylation profiling for cancer, neurodegenerative diseases, autoimmune diseases, and identification of specific glycan biomarkers (e.g., sialylated IgG glycosylation).
2. Drug Targets and Antibody Drug Development: Analyzing the effect of glycosylation on the Fc region of therapeutic antibodies (e.g., PD-1 monoclonal antibody) on antigen binding and ADCC efficacy, guiding engineering modification.
3. Pathogen-Host Interaction Mechanisms: Revealing modification patterns of viral glycoproteins (e.g., SARS-CoV-2 spike protein glycosylation shield) to aid vaccine design.
4. Cell Therapy Quality Control: Dynamic monitoring of glycoproteins on CAR-T cell surfaces to ensure product stability and efficacy.
How Can We Help You?
1. Communication of Requirements: Filling out the inquiry below to receive a customized solution.
2. Sample Submission: We support for dry ice shipping and accept samples like cells (>1×10^7), serum (>100 μL), and tissues (>50 mg).
3. Experiment and Analysis: Talilored analysis will be completed within 15 business days (routine projects), with real-time progress updates for complex projects.
4. Report Delivery: Including raw data, analysis results, and technical consultation support.
In the golden age of precision medicine and biopharmaceuticals, the deep analysis of glycosylation modifications has become the key to breaking research bottlenecks and developing groundbreaking therapies. MtoZ Biolabs provides a simple, efficient, and worry-free DIA-based glycoproteomics services that unlock the "hidden glycosignals" in trace samples. Contact our glycoscience experts today to take the lead in the cutting-edge field of glycoscience.
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