EZGlyco® O-Glycan Prep Kit
EZGlyco® O-Glycan Prep Kit provides a streamlined, rapid and reliable O-glycan sample preparation. The complete kit includes all reagents to release, enrich, label and purify O-glycans for HPLC and LC-MS analysis. The kit adopts a unique O-glycan releasing method using a highly reactive amine and an organic superbase, which yields high recovery and less degradation (peeling) of released O-glycans.
Applications include characterization of O-glycosylation of recombinant glycoproteins in upstream R&D, quality control and process control. It is also suitable for O-glycan biomarker discovery in biological samples such as mucins, serum and exosome, and O-glycan analysis of functional foods such as dairy products.
Advantages of EZGlyco® O-Glycan Prep Kit
- O-glycan Sample Preparation in 5 hours
O-glycan sample preparation is completed in 5 hours with its unique O-glycan release reagents. Thus, HPLC/LC-MS analysis is achieved on the same day. The kit contains 2-AB labeling reagent for a sensitive detection with LC and LC-MS analysis. - High Recovery
A combination of the unique O-glycan releasing reagent and O-glycan Capturing Beads enables high recovery of released O-glycans from various glycoproteins. - Minimized "Peeling"
Glycan releasing reagents minimize decomposition of O-glycans (peeling), resulting in O-glycan characterization with higher accuracy. - Easy and Safe Operation
With a simple protocol to follow, O-glycan sample preparation is completed without any special laboratory equipment.
Workflow
For HPLC and LC-MS-ready sample preparation
The streamlined operation can be carried out in 5 hours without any complicated processes and special laboratory instruments.
Comparison Data
Comparison of 2-AB labeled bovine fetuin O-glycan analysis prepared with 2 methods: EZGlyco® O-Glycan Prep kit vs Hydrazinolysis
| EZGlyco® O-Glycan Prep Kit provides comparable O-glycan recovery with lower peeling products and faster operation relative to hydrazinolysis. |
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Reference Data
Dose-dependency (Linearity)
- Varying amount of bovine fetuin (0.5 - 200 μg) was subjected to O-glycan preparation using EZGlyco® O-Glycan Prep Kit (N=1).
- 1 μL of the recovered solution containing O-glycans was analyzed using HILIC mode UHPLC.
| Excellent linear recovery over a wide sample range is achieved using EZGlyco® O-Glycan Prep Kit. |
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Reproducibility
- 20 μg of bovine fetuin was subjected to O-glycan preparation using EZGlyco® O-Glycan Prep Kit (N=3 x 3 days).
| A representative LC chromatogram of 2-AB-labeled O-glycans in bovine fetuin. |
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Intra-assay variability
| EZGlyco® O-Glycan Prep Kit demonstrates glycan profile CV < 5% and total recovery CV< 10%. |
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Performance Test
2-AB labeled bovine fetuin O-glycan analysis prepared with different sample preparation kits.
- 200 μg of bovine fetuin was subjected to O-glycan preparation using a) EZGlyco® O-Glycan Prep Kit, b) Product A (alkaline β-elimination), and c) Product B (alkaline β-elimination).
- Recovered O-glycans were analyzed using HILIC mode UHPLC.
| O-glycan total recovery and the suppression of “peeling” are superior with EZGlyco® O-Glycan Prep Kit. |
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Applications
O-glycan profiles in standard glycoproteins
- Each sample was dissolved in 10 μL of pure water and subjected to O-glycan preparation using EZGlyco® O-Glycan Prep Kit.
- 1 μL of the recovered solution containing O-glycans was analyzed using HILIC mode UHPLC.
| O-glycans were detected in all samples with a low peeling ratio (less than 10%). |
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O-glycan profiles in human serum
- 20 μL of human serum was dried using a centrifugal evaporator, the dried human serum was dissolved in 10 μL of pure water and subjected to O-glycan preparation using EZGlyco® O-Glycan Prep Kit.
- 1 μL of the recovered solution containing O-glycans was analyzed using HILIC mode UHPLC.
| Low sample amount of human serum is sufficient for O-glycan analysis with EZGlyco® O-Glycan Prep Kit. |
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Kit Components
- Protocol
- Glycan Release Reagent A
- Glycan Release Reagent B
- Glycan Capturing Beads
- Filter Column
- 2-Aminobenzamide
- Reducing Reagent
- Cleanup Column
Required Equipment, Labware, and Reagents
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- Acetic acid (AcOH), reagent grade*
- Acetonitrile (ACN), reagent grade*
- Methyl alcohol (MeOH), reagent grade*
- Filter Column
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- 2-Aminobenzamide
- Reducing Reagent
- 1.5-mL microcentrifuge tubes
- Pipette and tips for 1000, 200, and 20 μL
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- Heating block for use at 37°C and 50°C
- Vortex mixer
- Microcentrifuge (used at 500 and 3,000 x g)
*These products are also available at FUJIFILM Wako.
References
1. Kameyama A., et al., A practical method of liberating O-linked glycans from glycoproteins using hydroxylamine and an organic superbase, Biochem Biophys Res Commun. 513(1), 186-192 (2019)
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For research use or further manufacturing use only. Not for use in diagnostic procedures.
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