Permethylation Kit
Product Description
Application: This kit is used for permethylation of glycans. The permethylated stable glycan sialic acid is used for MALDI mass spectrometry analysis of acids and aids in linkage analysis studies. The kit can be used with glycans released from glycoproteins.
Description: This kit contains reagents for glycan permethylation. NOTE: This kit does not include methyl iodide/iodomethane (see Shipping section for details)
The number of samples can hold up to 96 samples.
Sample volumes up to 1 µg released glycans.
Appropriate samples any unlabeled purified glycans, released from glycoproteins by PNGaseF,
PNGase A, beta-elimination or hydrazinolysis.
Storage: Store at 2‑8°C.
Shipping: Product should be shipped between 2‑8°C.
NOTE: Due to shipping limitations of methyl iodide/iodomethane, we cannot provide this component with the kit. Therefore, we recommend purchasing iodomethane purity ≥99.0% (GC) from your local chemical supplier. Iodomethane Synonyms: Methyl iodide CAS No: 74‑88‑4
Safety: For Research Use Only. Not for human or medicinal use.
Kit Contents
The kit contains the following items:
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Note: This kit can be used to process 1-96 samples. The plate is expandable and can be used for any number of samples between 1 and 96. Store unused kit reagents and unused plate wells under recommended storage conditions for further sample preparation at a later date. Please use it within the validity period.
Additional reagents and equipment required
Methyl iodide (MeI) (synonym: methyl iodide) with ≥99.0% (GC) purity.
Pure water: resistivity higher than 18 M ‑cm, particle free (>0.22 µm), TOC <10 ppb.
Plate Vibrator
Reaction Eppendorf vials (1.5 or 2.0 ml) for final transfer of extracted samples (vials need to be resistant to chloroform or methylene chloride, otherwise use glass vials).
pH indicator strip
Centrifugal evaporator (eg Savant, HETO or similar).
70% methanol (MeOH)
Dichloromethane (DCM) (for adding to equilibration plate)
Program timeline
Manual permethylation of 96 samples takes approximately 5 to 6 hours.
Step 1: Permethylation of the sample:
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Step 2: Extraction and storage of permethylated samples
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Method Part 1: Permethylation of Glycans
a) Preparation of glycans
After N-glycans are released, we recommend users to enrich and purify N-glycans prior to permethylation to improve reaction efficiency. For this, we recommend using the 96 board. If desired, it can be purchased separately from . After filling,
Glycans need to be dried before they can be fully methylated. Dried glycans are best placed in 1.5 mL Eppendorf vials or similar. However, if you are using a 96 plate for enrichment and eluting the sample into a 96-well collection plate, you can dry the sample in the collection plate in preparation for the next step.
b) Add DMSO and dried glycan samples to permethylation plates
NOTE: Before starting the experiment, make sure the DMSO is completely thawed. DMSO is solid at 19°C.
Dissolve each glycan sample with 300 µL of DMSO. Vortex and centrifuge these samples. Peel the clear seal from the permethylated plate and cut off the overhang. Add the first sample in DMSO to the first well of the permethylation plate and use the same pipette tip to disrupt the well with the solid at the bottom. Repeat this process for the remaining samples and record the sample positions.
c) Incubation
Cover the plate with the provided 96-well silica plate cover, place the permethylated plate on a plate shaker at approximately 100‑150 rpm, and incubate at room temperature for 15 min.
NOTE: Cut the silicone 96-well cap/strip to cover the well containing the sample, making sure to securely cover the sample well.
After 15 minutes of incubation, briefly centrifuge the sample plate to ensure that all samples are at the bottom of the plate wells.
NOTE: Empty balance boards are provided as part of the kit. Ensure that the balance plate is properly equilibrated with the sample plate containing DMSO by adding water (equal volume weight).
d) Add MeI to the permethylation plate
Carefully remove the silica plate cover to avoid sample cross-contamination and add 55 µL MeI to the well containing the sample. Cover the wells again with the silicone plate caps, positioning them to cover the same sample wells as before and seal them securely.
e) Incubation
Make sure the silicone cap seals tightly to eliminate the loss of volatile MeI.
Place the permethylation plate securely on the plate shaker at 100‑150 rpm, taking care to observe that the sample contents on the 1.2 mL permethylation plate do not spill or carry over. Incubate the plate at room temperature for 60 minutes
After 60 minutes of incubation, briefly centrifuge the sample plate to ensure that all samples are at the bottom of the plate wells.
NOTE: Empty balance boards are provided as part of the kit. Ensure that the equilibration plate is properly equilibrated with the sample plate containing DMSO and MeI by adding water (equal weight by volume).
Method Part 2: Extraction of Permethylated Glycans
a) Extraction of permethylated glycans
Remove the silicon plate cover and add 450 µL of DCM to each sample well followed by ~500 µL of water in the permethylated plate.
To facilitate the extraction step, transfer the entire contents of each sample-containing well (DMSO, DCM, and water) into a labeled 1.5/2.0 mL Eppendorf vial or glass vial.
After transferring the samples, mix the organic DCM layer and aqueous layer of each sample by vortexing. Place the vials on the rack, allowing the two layers to separate.
Discard the upper aqueous layer into a chlorinated waste container.
Add 800 μL of water to each sample and wash the organic layer again. Vortex each sample to ensure good solvent mixing and allow the two layers to separate.
Discard the upper aqueous layer into a chlorinated waste container.
Repeat the washing step with an additional 800 µL of water and test with pH paper until the aqueous layer is no longer alkaline (if > 7, repeat the washing until the pH of the aqueous layer is ≤ 7).
b) Drying of permethylated glycans
Make sure to completely remove the top aqueous layer present in each vial and discard into a chlorinated waste container. Permethylated glycans are present in the organic DCM layer. Dry the organic solvent containing the permethylated sample in a centrifugal evaporator.
NOTE: If liquid-liquid extraction is performed in a 96-well plate format, use the empty equilibration plate provided for the equilibrated centrifugal evaporator. Ensure that the equilibrated plate is properly equilibrated by adding DCM or chloroform (equal volume by weight) compared to the sample plate containing permethylated glycans in DCM.
NOTE: Once the sample plate and equilibration plate are placed in the centrifugal evaporator, spin the plate for 2 min without vacuum to allow the DCM to settle. After 2 minutes, the vacuum pump can be turned on to dry the permethylated glycans.
c) Storage of samples
Add 10 µL of 70% MeOH and dried permethylated glycans to each vial and mix by vortexing. Samples can be stored at –20°C prior to mass spectrometry analysis.
References
1.A. Ki‑Irem; GV Avakumov; IV Sidorova and A. Strel Chyonok (1979) "Methylation Analysis in Glycoprotein Chemistry" Chromatography 180: 69‑82
2. Dale; AJ Ration; Kay‑Hooi Khoo; M. Panico; A. McDowell and HR Morris (1994) "Mass Spectrometry of Carbohydrate-Containing Biopolymers", Methods in Enzymology 230: 108‑132
3.I. Chucanu; CE Costello (2003) "Elimination of Oxidative Degradation During Carbohydrate Peroxymethylation", J. Am. Chemical. Socialist Party. 125: 16213‑16219
4. I. Chucanu; (2006) "Per-O-methylation of carbohydrates for structural analysis by mass spectrometry", Acta Analytica Chem. 576: 147-155
5.I. Chucanu; R. Caprita (2007) "Peroxymethylation of neutral carbohydrates directly from water samples for gas chromatography and mass spectrometry", Analytica Chimica Acta 585: 81 ‑85
6. Shubhakar, A., Kozak, RP, Reiding, KR, Royle, L., Spencer, DIR, Fernandes, DL, Wuhrer, M.: Automated high-pass for glycosylation analysis of biological products using MALDI‑TOF MS amount of permethylation. analytical chemistry. acs.analchem.6b01639 (2016).