Glycosylation is one of the most common and complex post-translational modifications (PTMs) of proteins, particularly N-linked glycosylation (N-glycosylation), which is widely present in secreted and membrane-bound proteins. Minor variations in N-glycan structures can lead to significant changes in protein function, stability, immunogenicity, and biological activity. Therefore, accurate detection and analysis of N-glycans are of great importance in biopharmaceutical development, biomarker research, disease mechanism exploration, and quality control.
The Pro A N-glycan kit is designed for the rapid release and labeling of protein N-glycans and is applicable to glycosylation analysis in various fields, including biopharmaceuticals. This kit employs PNGase F to release N-glycans and uses a ProA labeling reagent for fluorescent tagging of glycans, enabling their detection by high-performance liquid chromatography (HPLC) and mass spectrometry (MS).
Product Overview
The Pro A N-glycan kit launched by MtoZ Biolabs is a sample pretreatment kit that integrates efficient N-glycan labeling, signal enhancement, and broad platform compatibility. This product utilizes an optimally designed Pro A labeling molecule that exhibits excellent fluorescence properties and significantly enhances mass spectrometry ionization efficiency. It is compatible with a variety of analytical platforms, including UPLC-FLD and HILIC-LC-MS/MS. Whether you are engaged in basic research or drug quality control, this kit provides robust support for your N-glycan analysis.
Product Details
|
Product Details |
Size |
Storage Conditions |
|
PNGase F |
100 µL/vial |
2-8℃ |
|
Digestion Buffer (7.5×) |
200 µL/vial |
2-8℃ |
|
Enhanced Digestion Buffer (7.5×) |
200 µL/vial |
2-8℃ |
|
Pro A Solution |
500 µL/vial |
-20℃ |
|
Reducing Agent Solution |
500 µL/vial |
-20℃ |
|
Purification Magnetic Beads |
10 mL/bottle |
2-8℃ |
|
Elution Buffer |
2 mL/vial |
2-8℃ |
|
Wash Buffer |
40 mL/bottle |
2-8℃ |
Protocol
The entire labeling workflow has been optimized by the MtoZ Biolabs team for routine laboratory procedures. The main steps are as follows:
1. Sample Digestion
This kit includes two enzymatic digestion approaches: non-denaturing digestion and denaturing digestion. The primary distinction lies in that certain N-glycan sites are difficult to cleave under non-denaturing conditions, thus requiring enhanced digestion buffer for effective release.
(1) Non-denaturing Digestion
Dilute the sample with water to 5 mg/mL. Take 24 µL of the diluted sample, add 4 µL of digestion buffer, then add 2 µL of PNGase F. Mix thoroughly and incubate at 50°C for 5 minutes for digestion, followed by denaturation at 98°C for 5 minutes. Centrifuge at ≥14,000 rpm for 3-5 minutes and transfer 25 µL of the supernatant into a new EP tube.
(2) Denaturing Digestion
Dilute the sample with water to 5 mg/mL. Take 24 µL of the diluted sample, add 4 µL of enhanced digestion buffer, mix well, and heat at 98°C for 3 minutes to denature. Then add 2 µL of PNGase F, mix thoroughly, and incubate at 50°C for 5 minutes. Follow with a denaturation step at 98°C for 5 minutes, and centrifuge to bring the sample to the bottom of the tube once it returns to room temperature.
2. ProA Labeling
While centrifuging, simultaneously prepare the labeling solution. Mix Pro A solution, reducing agent solution, and formic acid solution in a 2:2:1 volume ratio to prepare the labeling solution. After thorough mixing, take 25 µL of the labeling solution and add it to the previously obtained 25 µL supernatant or 30 µL digestion solution. Mix well and incubate at 50°C for 15 minutes. The volume of the labeling solution can be adjusted accordingly based on the number of samples.
3. Magnetic Bead Purification
(1) Binding
Take out the magnetic beads for purification, vortex to mix thoroughly, then pipette 200 µL of the bead suspension into an EP tube. Place the tube on a magnetic stand and, once the solution becomes clear, carefully remove the supernatant.
Add 385 µL of acetonitrile and pipette 3-4 times to mix thoroughly.
(2) Washing
Place the EP tube on the magnetic stand. After about 30 seconds, once the solution has cleared, carefully remove the supernatant. Remove the EP tube from the magnetic stand, add 400 µL of wash buffer, and pipette to mix thoroughly. Return the EP tube to the magnetic stand, wait about 30 seconds for the solution to clear, then discard the supernatant.
Repeat the above washing step once.
(3) Elution
Remove the EP tube from the magnetic stand, add 40 µL of elution buffer, pipette to mix thoroughly, and incubate at room temperature for 2-3 minutes. Place the tube on the magnetic stand. After about 30 seconds, once the solution has cleared, carefully transfer 40 µL of supernatant to a new EP tube and add 60 µL of acetonitrile to mix. Centrifuge at ≥14,000 rpm for 3 minutes and transfer the supernatant into a sample vial.
Note: It is recommended to add liquid along the inner wall of the tube above the magnetic beads to help wash the beads down to the bottom smoothly.
Features and Benefits
1. Dual-Mode Enhancement for Fluorescence and Mass Spectrometry
The Pro A tag is designed to enhance both fluorescence detection and mass spectrometry response, making it compatible with multiple analytical platforms. One labeling step, multiple applications.
2. High Labeling Efficiency with Low Background Interference
The reaction exhibits high efficiency with minimal side reactions, reducing non-specific signals and ensuring accurate downstream quantification.
3. Mild Reaction Conditions with Broad Sample Compatibility
The labeling reaction proceeds under gentle conditions, making it suitable for free N-glycan samples derived from various sources.
4. High Stability for Long-Term Storage and Batch Analysis
The labeled products demonstrate excellent stability, allowing for refrigerated storage prior to analysis and facilitating experimental scheduling and quality control.
Applications
The Pro A N-glycan kit can be applied in a variety of research and application fields, including but not limited to:
1. Glycosylation Analysis of Biopharmaceuticals
Used for structural characterization and quality consistency evaluation of N-glycans in biopharmaceuticals such as monoclonal antibodies (mAbs) and fusion proteins.
2. Disease-Associated Glycomics Research
Investigates changes in N-glycan expression in diseases such as cancer, inflammation, and infections to identify potential biomarkers.
3. Vaccine and Glycoantigen Development
Analyzes glycan structures on the surface of viruses, bacteria, or parasites to support vaccine design and functional validation.
4. Natural Products and Glycoengineering
Explores the structure-activity relationship of carbohydrate compounds derived from plants or microorganisms for screening novel functional glycans.
5. Glycosyltransferase Function Validation and Substrate Screening
Combines with in vitro glycosylation reactions to monitor glycan changes in enzymatic products and improve substrate screening efficiency.
FAQs
Q1: Are Pro A-Labeled N-Glycans Compatible with Both LC-MS and UPLC-FLD Platforms?
A1: Yes, the Pro A labeling reagent provides dual enhancement for fluorescence detection and mass spectrometric response, making it compatible with both platforms.
Q2: Is the Kit Suitable for Low-Abundance Flycan Samples?
A2: Yes. The kit features high labeling efficiency and signal amplification, which enhances detection sensitivity for low-abundance samples.
Q3: Is Further Purification Needed after Labeling?
A3: If mass spectrometry is used for downstream analysis, a simple desalting step or SPE column purification is recommended to remove residual contaminants.
Q4: Can Labeled Samples Be Stored for Later Analysis?
A4: Yes. It is recommended to store labeled products at -20°C, avoid repeated freeze-thaw cycles, and perform analysis within two weeks for optimal results.
