Sample Preparation Tip
Level: Intermediate
Sample Prep for N-linked Glycans
The use of a sample prep plate for cleaning up N-linked glycans cleaved from glycoproteins
Sample preparation plates have proven successful in the replacement of more intensive sample preparation processes which follow glycan cleavage from glycoproteins using PNGase F digestion. It is essential to label the N-glycans prior to quantitation by HPLC-UV or LC-MS/MS; these more traditional clean up methods are time consuming and contain multiple steps which can increase error. Clean up using an HILIC solid phase extraction (SPE) product, such as Biozenâ„¢-N-Glycan Clean-Up microelution plates, offers sensitive, effective, timely results in comparison. We outline the glycan workflow below.
Intact Glycoprotein
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Glycoprotein samples are reconstituted to 2mg/mL with pure water and denatured by adding 6µL of surfactant and heating to 90°C for 3 minutes. 1.2µL of PNGase F is added and samples incubated for 5 minutes at 50°C.
This step serves to cleave the glycans from their glycoprotein.
Deglycosylation with PNGase F
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Intact Glycoprotein
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Labelling is then done using either 2-amino acetic acid (2-AA) or 2-aminobenzoic acid (2-AB) using the released glycans which are prepared with 12 μL of labelling reagent solution per sample and incubated for 5 minutes at room temperature.
After fluorescent labeling, the dye must be removed as this is in excess as is common in any derivatization step. For this an HILIC SPE plate is preferred.
Sample with excess dye; Then HIILIC sample prep (silica NH2)
| 96-Well Plate | Biozen N-Glycan Clean-Up |
|---|---|
| Condition | 200µL Water |
| Equilibrate | 200µL Water /ACN (15:85) |
| Load | Sample |
| Wash | 2 x 600µL Formic acid / water / ACN (1:9:90) |
| Elute | 3 x 30µL 200mM ammonium acetate in ACN/water (5:95) |
| Dilute | 100µL Dimethylforamide and 210µL ACN |
This step elutes the glycans in aqueous buffer, however their HPLC separation technique is commonly HILIC and an aqueous diluent would constitute a strong solvent for a HILIC method, making it less than ideal. For this reason, micro elution plates are the preferred platform for the clean up, to minimize the amount of aqueous buffer in the sample and reduce the strong solvent effect for subsequent analysis. When prepared in this way, these labelled glycan samples are ready for analysis using a Glycan analysis column.
Sample Clean-Up After N-Linked Glycan Release and Labeling Using Biozen N-Glycan Clean-Up HILIC SPE Microelution 96-Well Plate
This application note compares two glycan clean-up solutions for a commercially available in-solution digestion and labeling kit for glycan profiling of a trastuzumab biosimilar and a heavily sialylated glycoprotein (alpha-1 acid glycoprotein, AGP). Biozen N-Glycan Clean-Up is a HILIC solid phase extraction product in a microelution 96-well plate that has excellent retention and recovery of labeled, released n-glycans.
