What types of glycosylation can you characterize?

We identify and map N-linked and O-linked glycosylation sites using a combination of enzymatic releases (PNGase F, O-glycosidase) and LC-MS/MS glycopeptide analysis. The glycan types characterized—including high mannose, complex, hybrid, and sialylated forms—are scoped per project based on your product and expression system.

What sample amount is needed for analysis?

Typical projects require 50–200 μg of purified protein for peptide mapping and intact mass analysis. Exact amounts are determined during the project scope consultation and depend on the desired depth of characterization.

How do you handle data analysis for complex glycopeptide spectra?

We use specialized software such as Byonic, Protein Metrics, or GlycoQuest combined with manual verification by experienced scientists. Glycan assignments are supported by accurate mass, fragmentation spectra, and retention-time matching to standards when available.

Can you analyze heavily sialylated or anionic glycoproteins?

Yes, our methods are optimized for neutral and acidic glycan forms. We use appropriate LC conditions (e.g., HILIC with low-pH mobile phases) and MS detection to minimize desialylation and ensure accurate profiling.

Do you provide regulatory-compliant reports?

Our reports follow industry best practices for CMC documentation, including method descriptions, raw data archives, and summary tables. Regulatory strategy support can be customized as part of the project scope.

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Glycoprotein Characterization and Stability Analysis

Glycoprotein Characterization

Comprehensive LC-MS/MS-based profiling of glycosylation, PTMs, and higher-order structure to support biologic development and quality control.

LC-MS/MS peptide mapping for sequence verification and PTM identification
N- and O-linked glycosylation mapping with site occupancy determination
Intact mass analysis for glycoform distribution and molecular weight confirmation
Higher-order structure analysis via CD, FTIR, DSC, and AUC
Stability and degradation product profiling including aggregation and fragmentation

Speak with an Expert

Why Is Glycoprotein Characterization Critical for Your Biologic?

Glycosylation is a critical quality attribute that affects stability, efficacy, and immunogenicity of therapeutic proteins.

Site-specific identification of N- and O-glycans
Quantitative PTM mapping (oxidation, deamidation, glycation)
Intact mass and subunit mass analysis for product heterogeneity
Complementary biophysical and binding assays
Expert data interpretation and regulatory-compliant reporting

Reproducible

Comprehensive

Regulatory-ready

Our Approach

Integrated Glycoprotein Characterization Workflow

Combining mass spectrometry-based peptide mapping with biophysical and activity assays to provide a complete picture of your protein's structural and functional properties.

LC-MS/MS Peptide Mapping & PTM Analysis

Our core platform uses high-resolution LC-MS/MS after enzymatic digestion to achieve deep sequence coverage and pinpoint post-translational modifications including glycosylation.

Trypsin and multiple protease digests for >95% sequence coverage
PNGase F treatment for N-glycan release and site assignment
CID/HCD fragmentation for glycosylation site localization
Quantitation of oxidation, deamidation, glycation levels

Higher-Order Structure & Functional Characterization

Complementary biophysical techniques and activity assays confirm that glycosylation does not compromise protein folding or bioactivity.

Circular dichroism (CD) and FTIR for secondary structure
DSL and AUC for aggregation and particle size
SPR-based binding affinity for receptor/antigen interaction
Enzymatic activity assays for functional integrity

Quality & Reproducibility

Rigorous sample preparation protocols and system suitability standards ensure batch-to-batch consistency across projects.

Statistical Rigor

Data analysis includes automated glycan assignment with false-discovery control and replicate analysis for quantitative confidence.

Biological Interpretability

Results are placed in the context of known structure-activity relationships to guide formulation, process, and comparability decisions.

Deliverables

What You Receive

Comprehensive data packages designed to support regulatory submissions and internal decision-making.

Data Deliverables

Raw LC-MS/MS data files (Thermo.raw or equivalent)
Processed peptide maps with annotated PTMs
Glycan assignment tables (compositions, relative abundance, site occupancy)
Intact mass spectra with deconvoluted masses
Biophysical characterization data (CD, FTIR, DSC, AUC)
Quantitative degradation and impurity profiles

Reporting & Support

Full study report with methods, results, and interpretation
Summary slide deck for internal review
On-site or virtual data review meeting
Regulatory-support documentation (methods, validation summaries as scoped)
Ongoing scientific consultation throughout project

Service Scope

Customizable Characterization Parameters

Each project is scoped around your specific molecule and development stage. Typical parameters include:

Parameter	Typical Project Scope
Sample requirements	50–200 μg purified protein (adjusted per scope)
Digestion strategy	Trypsin, Glu-C, Lys-C, PNGase F, O-glycosidase
LC-MS instrumentation	Orbitrap-based HRMS (Thermo Q Exactive, Fusion Lumos)
Sequence coverage target	≥95% with optimized digestion conditions
Glycan types analyzed	N-linked, O-linked; high mannose, complex, hybrid, sialylated
PTM quantitation	Oxidation, deamidation, glycation (relative % per site)
Biophysical assays	CD, FTIR, DSC, DLS, AUC, SPR (selected per project)
Data analysis	Byonic, Protein Metrics, GlycoQuest, manual verification
Deliverables format	PDF study report, raw data archives, slide summary

How It Works

Streamlined Project Workflow

From sample receipt to final report, each step is managed by experienced scientists with clear communication.

Sample Receipt and Preparation

Protein samples are buffer-exchanged, quantified, and subjected to enzymatic digestion (trypsin, Glu-C, or PNGase F) under controlled conditions to generate peptides for analysis.

LC-MS/MS Data Acquisition

Peptide mixtures are separated by reverse-phase chromatography and analyzed on high-resolution Orbitrap mass spectrometers to generate high-quality fragmentation spectra.

Peptide Mapping and PTM Assignment

Database search against the protein sequence identifies peptides and maps modifications including glycosylation, oxidation, and deamidation using validated software.

Glycan Profiling and Intact Mass Analysis

Released glycans are analyzed by HILIC-FLR and/or LC-MS; intact protein is analyzed for glycoform distribution and molecular weight confirmation.

Reporting and Review

All data are compiled into a comprehensive report with annotated spectra, quantitation tables, and expert interpretation from our protein characterization team.

Applications

Tailored for Diverse Biologic Modalities

Our glycoprotein characterization platform supports a wide range of recombinant proteins, antibodies, and viral vectors.

mAb Glycosylation Profiling

Characterize N-glycosylation patterns at Fc and Fab domains to monitor manufacturing consistency and assess biosimilarity.

Fc glycan profiling (G0F, G1F, G2F, high mannose, sialylation)
Fab glycosylation when present
Hotspot PTM monitoring (oxidation, deamidation on CDRs)
Comparability studies for biosimilar development

Recombinant Glycoprotein Characterization

Full structural and PTM characterization of enzymes, cytokines, and fusion proteins produced in CHO, HEK, or other expression systems.

Site-specific N- and O-glycan mapping
Intact mass and subunit analysis
Aggregation and fragmentation profiling
Stability-indicating assay development

Viral Glycoprotein Analysis

Peptide mapping of viral envelope and capsid proteins to verify identity and glycosylation critical for immunogenicity.

Capsid protein sequence coverage and PTM mapping
Glycan site occupancy on attachment proteins
Lot-to-lot consistency for process validation
Support for regulatory filings

FAQ

Frequently Asked Questions

Common questions about our glycoprotein characterization services.

We identify and map N-linked and O-linked glycosylation sites using a combination of enzymatic releases (PNGase F, O-glycosidase) and LC-MS/MS glycopeptide analysis. The glycan types characterized—including high mannose, complex, hybrid, and sialylated forms—are scoped per project based on your product and expression system.
Typical projects require 50–200 μg of purified protein for peptide mapping and intact mass analysis. Exact amounts are determined during the project scope consultation and depend on the desired depth of characterization.
We use specialized software such as Byonic, Protein Metrics, or GlycoQuest combined with manual verification by experienced scientists. Glycan assignments are supported by accurate mass, fragmentation spectra, and retention-time matching to standards when available.
Yes, our methods are optimized for neutral and acidic glycan forms. We use appropriate LC conditions (e.g., HILIC with low-pH mobile phases) and MS detection to minimize desialylation and ensure accurate profiling.
Our reports follow industry best practices for CMC documentation, including method descriptions, raw data archives, and summary tables. Regulatory strategy support can be customized as part of the project scope.

Scientific Literature

References

Feng Y, Li Y, Zhang Z, et al. Characterization and functional analysis of N-linked glycosylation on the Hendra virus attachment glycoprotein. Virology journal. 2026;23(1). View on PubMed
Liu Q, Lu X, Deng Y, et al. Global characterization of mouse testis O-glycoproteome landscape during spermatogenesis. Nature communications. 2025;16(1):2676. View on PubMed
Delobel A. Glycosylation of Therapeutic Proteins: A Critical Quality Attribute. Methods in molecular biology (Clifton, N.J.). 2021;2271:1-21. View on PubMed
Kumar P, Thakur R, Acharya PC, et al. Synthesis, characterization, and radiosynthesis of fluorine-18-AVT-011 as a Pgp chemoresistance imaging marker. Scientific reports. 2022;12(1):18584. View on PubMed
Ou X, Wang H, Tie H, et al. Novel plant-derived exosome-like nanovesicles from Catharanthus roseus: preparation, characterization, and immunostimulatory effect via TNF-α/NF-κB/PU.1 axis. Journal of nanobiotechnology. 2023;21(1):160. View on PubMed
Veith PD, Shoji M, Scott NE, et al. Characterization of the O-Glycoproteome of Porphyromonas gingivalis. Microbiology spectrum. 2022;10(1):e0150221. View on PubMed

Please kindly note that our products and services are for research use only.