N-glycosylation Analysis of Proteins

Q: What are the typical biological samples compatible with your N-glycosylation analysis service?

Creative Biolabs conducts routine analysis of numerous biological samples which range from purified recombinant proteins derived from mammalian, yeast, insect or plant systems to serum/plasma samples and intricate biological fluids as well as membrane-enriched fractions. Our research capabilities extend to site-specific glycan profiling of proteolytically digested glycoproteins and we provide enrichment methods for low-abundance glycoproteins through lectin affinity or hydrazide chemistry. Our workflows are customizable for analytical depth and sensitivity requirements regardless of your work with antibodies, therapeutic enzymes or virus spike proteins.

Q: How do you analyze complex-type N-glycans and distinguish sialylation or fucosylation patterns?

We use a combination of HILIC-UPLC coupled with fluorescence detection and MALDI-TOF-MS or ESI-MS/MS to resolve complex-type N-glycans with high precision. Structural features such as terminal sialic acid linkages (α2,3 vs α2,6) and fucosylation (core vs antennary) are determined through exoglycosidase digestion arrays, linkage-specific derivatization, or tandem MS. For absolute differentiation, we incorporate permethylation or reductive amination prior to analysis. These methods are particularly useful when profiling glycosylation changes in disease-associated proteins or therapeutic biologics.

Q: What is the minimum protein amount required for N-glycan analysis?

For released N-glycan profiling via fluorescence-assisted HILIC or MALDI-MS, as little as 5–10 µg of glycoprotein is sufficient. For intact glycopeptide mapping by LC-MS/MS, we typically recommend 50–100 µg depending on protein complexity and desired coverage. If you have limited sample, we offer miniaturized workflows and sensitivity-enhanced strategies such as RapiFluor-MS labeling or nanoLC-MS.

Introduction Workflow Techniques Services FAQs Products Supports

N-linked glycosylation and O-linked glycosylation are both ubiquitous and essential post-translational modifications. N-linked glycosylation refers to the attachment of oligosaccharide chains to the nitrogen atom of asparagine residues in proteins. It begins in the ER with the en bloc transfer of a lipid-linked oligosaccharide (Glc3Man9GlcNAc2) to nascent polypeptides at the Asn-X-Ser/Thr consensus motif.

Fig.1 Structural Characterization of Human Plasma N-Glycans. (OA Literature) Fig.1 N-glycosylation landscape of human plasma proteins.¹

Process in the ER and Golgi

In the ER:
- Assembly: Carried out by ALG gene products onto Dolichol phosphate carriers.
- Trimming & Folding: α-glucosidases I and II remove terminal glucose, which facilitates protein folding by allowing interaction with calnexin/calreticulin chaperones.
- This chaperone cycle ensures proper folding, and misfolded proteins are retained or degraded.
In the Golgi:
- Processing into subtypes: Complex, hybrid, or high-mannose forms via trimming and extension of the core glycan (GlcNAc₂Man₃).
- Further additions include fucose, sialic acid, galactose, leading to structural heterogeneity critical for protein function and trafficking.

Biological Importance

N-glycosylation directly stabilizes protein tertiary structure and indirectly aids through lectin-mediated chaperone recognition.
Acts as a tag for the ER-associated degradation (ERAD) pathway if folding fails.
Influences sorting and secretion of proteins, including immune receptors and enzymes.

Benefits of N-linked Glycosylation

Functional Benefit	Mechanism/Impact
Protein folding & stability	Via ER chaperones (calnexin/calreticulin), stabilization of folding intermediates
Intracellular trafficking	Ensures ER-to-Golgi transition and efficient membrane targeting
Immunogenic modulation	Specific glycan motifs regulate immune recognition
Therapeutic half-life extension	Alters serum clearance rates of therapeutic proteins

Step-by-Step Workflow for N-Linked Glycosylation Analysis

1. Protein Preparation & Enrichment

Accepted samples include cell lysate, secreted proteins, purified recombinant glycoproteins.
Enrichment is an optional but common step before analysis. We use lectin affinity (e.g., ConA for high mannose), hydrazide chemistry, or HILIC (hydrophilic interaction liquid chromatography) for glycopeptide/glycoprotein capture.

2. Glycan Release

Enzymatic method is commonly used in this section while chemical method is rarely used due to harshness
Use PNGase F to release N-glycans from Asn-X-Ser/Thr motifs.
PNGase A can be used if α1,3-fucose is present (e.g., in plant glycoproteins).

3. Glycan Labeling (Optional for Enhanced Detection)

Improves fluorescence and MS ionization.
Common fluorescent tags:
- 2-AB (2-aminobenzamide)
- Procainamide, 2-AA, or PA (pyridylamination)

4. Separation Techniques

A. Released Glycans

HILIC-UPLC or CE with FLR detection for glycan profiling.
MALDI-TOF-MS or LC-MS/MS for mass determination and structural info.

B. Glycopeptides

Perform proteolysis (e.g., trypsin) on native or deglycosylated protein.
Analyze by LC-MS/MS for:
- Glycosite occupancy
- Glycan heterogeneity per site
- Peptide backbone info

5. Structure Elucidation

Use exoglycosidase arrays or MS/MS fragmentation to determine branching, terminal residues (e.g., sialic acid, fucose), and linkage positions.
For deep analysis, combine:
- MSn (multiple-stage fragmentation)
- NMR spectroscopy (for linkage/conformation confirmation)
- Glycan sequencing tools

6. Data Analysis Tools

Glycopeptide identification
Predict glycan compositions from MS data
Annotate and visualize glycan structures
Quantitative MS analysis of glycopeptides

7. Quantification

Relative: Based on peak areas from FLR or MS signal intensities.
Absolute: Use glycan standards or stable isotope-labeled internal standards.

Analytical Techniques for N-Glycosylation

HILIC-FLR-MS is widely used in industrial QC pipelines for its balance of throughput, resolution, and MS compatibility.
LC-MS/MS of glycopeptides is the gold standard for site-specific glycosylation analysis.
CE-LIF is a regulatory-approved method (e.g., for biosimilars), especially in Europe and Asia.
MALDI-MS is often used as a screening tool or in early-stage glycan discovery.
More techniques for N-glycosylation and their pros and cons are as followed:

Technique	Analyte	Detection	Strengths	Limitations	Typical Applications
HILIC-FLR/UV-MS	Released glycans	Fluorescence/MS	High resolution, quantifiable, compatible with MS	Requires derivatization (e.g., 2-AB, RFMS); cannot localize site	Profiling glycan heterogeneity in mAbs and glycoproteins
MALDI-TOF-MS	Released glycans	MS only	Fast, sensitive, minimal sample needed	Limited quantitation; isomer resolution poor	Rapid glycan mass profiling; glycoform distribution
CE-LIF	Released glycans	Laser-induced FLR	High separation efficiency, low sample amount	Specialized equipment; limited structural info	High-throughput glycan profiling; QA/QC for biologics
LC-MS/MS (Bottom-up)	Glycopeptides	ESI-MS/MS	Site-specific glycosylation, glycan + peptide info	Complex data analysis; signal suppression from glycan moiety	Mapping glycosites, studying microheterogeneity
Top-down MS / Middle-down MS	Intact glycoproteins	ESI-MS	Intact glycoform distribution, charge envelope visualization	Requires ultrahigh-res instruments; lower sensitivity	Intact glycoprotein characterization; mAb Fc glycan profiling
Glycan Array (Lectin-based)	Native glycans	Fluorescence	Functional glycan-binding assay	Indirect; no structural info or quantification	Glycan-binding studies (e.g., Siglec or lectin profiling)
Exoglycosidase Digestion + MS	Released glycans/glycopeptide	MS/MS	Linkage, branching elucidation	Labor-intensive, sequential steps	Structural elucidation; verifying sialylation/fucosylation
NMR Spectroscopy	Pure glycans	NMR (¹H, ¹³C)	Linkage and anomeric configuration; unambiguous structure	Requires large amount of purified glycans; low sensitivity	Confirming full glycan structure; reference standard creation

What We Offer

N-linked glycosylation governs a wide array of biological processes, and its dysregulation underlies various human diseases—from congenital glycosylation disorders and autoimmune diseases like rheumatoid arthritis, to tumor progression and viral pathogenesis. At Creative Biolabs, we offer a full suite of customizable N-glycosylation research services to help you decipher glycan-related mechanisms and translate them into therapeutic opportunities.

✔ Glycosylation Profiling & Structural Characterization

Our high-resolution LC-MS/MS and HILIC-FLR platforms enable accurate mapping of N-glycan structures, glycosite occupancy, and branching patterns in both native and recombinant glycoproteins.

✔ Site-Specific Glycopeptide Analysis

We specialize in site-resolved glycoproteomics to uncover disease-specific glycan alterations—ideal for biomarker discovery in cancer and autoimmune diseases.

✔ Therapeutic Glycoprotein Engineering

Using glycoengineering strategies (e.g., CHO Lec mutants, FUT8 knockout, ENGase-mediated glycan remodeling), we help clients design recombinant antibodies, Fc-fusion proteins, or enzymes with optimized N-glycoforms for enhanced pharmacological performance.

✔ Anti-Glycan Immunotherapy Development

We offer custom services to generate anti-glycan antibodies, glycan-conjugated vaccines, and tumor-associated carbohydrate antigen (TACA) screening platforms—empowering glycan-targeted cancer immunotherapy.

FAQs

What are the typical biological samples compatible with your N-glycosylation analysis service?

How do you analyze complex-type N-glycans and distinguish sialylation or fucosylation patterns?

What is the minimum protein amount required for N-glycan analysis?

Reference:

Clerc, Florent, et al. "Human plasma protein N-glycosylation." Glycoconjugate journal 33 (2016): 309-343. Distributed under Open Access license CC BY 4.0, without modification. https://doi.org/10.1007/s10719-015-9626-2