V-Tag Glycopeptide Labeling Kit
Specifications for LT-VTAG-24
Application：For the fluorophore labeling and enrichment of glycopeptides.
Description：This kit contains the reagents needed for the conjugation of V-tag dye (LT- VTAG-01) to
the amine moieties of glycopeptides, and the specialised solid phase extraction (SPE)
cartridges required for the purification and enrichment of the labeled glycopeptides.
Dye Properties：Mass = 434.42 (resulting in a glycopeptide mass increase of 319.33 Da.) Fluorescence, λex= 250 nm,λem= 360 nm.
Number of Samples：24 separate analytical samples per kit.
Amount of Sample：From approximately 1 µg to 50 µg of glycoprotein per sample.
Suitable Samples：IgG single subclass glycoproteins. Other glycoproteins can be used, but note that if
there are multiple glycosylation sites, the glycopeptides may need to be separated by
C18 HPLC before HILIC analysis. It is recommended that samples do not have amine
containing solvents (e.g. Tris type buffers) or reduction/alkylation agents as they will
interfere with the V-tag labeling.Removal of the amine containing solvents and
reduction/alkylation agents can be accomplished using devices which are designed for
desalting and buffer exchange, as well as removal of low-molecular weight compounds.
Labeling Selectivity：One V-tag label for every IgG glycopeptide N-terminus.
Storage：Store the V-tag dye at -20°C in the dark. Protect from sources of heat, light and
moisture. Once used, extra dye solution can be frozen and re-used.
Shipping:The product can be shipped at ambient temperature
Handling: Ensure that any glass, plastic-ware or solvents are free from unwanted peptidases,
glycosidases and environmental carbohydrates. Use powder-free gloves for all sample
handling procedures and avoid contamination with environmental carbohydrates.
Safety: For research use only. Not for human or drug use.
Additional Reagents and Equipment Required
Heating block, oven, or similar dry heater set at 37°C for labeling reaction
Reaction vials (e.g. polypropylene micro-centrifuge vials)
Collection vials (e.g. polypropylene micro-centrifuge vials) or 96-deep well plate
Pipettes (1 to 10 µL, 10 to 100 µl, 20 to 200 µl capacity and 100 to 1000 µl capacity)
Vortex and centrifuge
Acetonitrile (LC/MS grade)
18.2 MΩ·cm Water
96-well plate extraction vacuum manifold and vacuum pump (optional)
Time Line for Labeling
V-tag labeling and enrichment of glycopeptides method takes approximately 2.5 hours:
Procedure | Time |
Preparation samples | 5 min |
Addition of dye to samples | 5 min |
Incubate samples | 1 hour |
Clean up | 1 hour |
Labeling Reaction
1，Defrost the V-tag dye solution
Gently defrost it at room temperature before use.Once the V-tag dye is used the spare solution can be re-frozen and re-used. As with many fluorescent dyes, care should be taken to minimise exposure to light as it will degrade the dye over time.
2，Add V-tag dye to samples
Add 5 μL of the V-tag dye directly to each enzyme digested sample. Vortex and briefly centrifuge thesamples.
3，Incubate
Place the reaction vials in a heating block or dry oven set at 37°C and incubate for 1 hour.
4， Centrifuge and cool
After the incubation period, briefly centrifuge the micro-tubes and allow them to completely cool to room temperature.
5， Prepare the washing solutions
Prepare solutions 1, 2, and 3 using 10% TFA, acetonitrile and water
NOTE FOR SOLUTIONS 2 and 3: These solutions should be prepared by measuring the volumes of water, acetonitrile and TFA independently and accurately before combining together. The composition of these solutions is critical for good enrichment results.
Solution 2.Mix together: 152 mL ACN, 46 mL water and 2 mL of the 10% TFA solution.
Solution 3.Mix together: 40 mL ACN, 59 mL water and 1 mL of the 10% TFA solution.
6，Prepare the LC-A cartridges
Place a LC-A cartridge for each sample into the cartridge holder, and position onto a vacuummanifold.
Prime each LC-A cartridge by adding the following solutions, applying a slow vacuum to drain and discarding the flow-through.
7，Prepare the glycopeptide samples and apply to the LC-A cartridge
Pipette 150 µL of 100% acetonitrile into the fluorophore labeled sample (typically the volume of the fluorophore labeling mix + glycopeptide sample is 15-25 μL). Gently mix the sample by pipette action and immediately load each sample onto a primed cartridge. Wait 5 minutes and then apply a slow vacuum (taking approximately one minute) to drain the LC-A cartridge.
Note: In order to avoid sample precipitation, the addition of acetonitrile should be performed just before applying the sample onto the cartridge.
8，Wash the LC-A cartridges
Wash the cartridges with 3 x 1 mL of 76% acetonitrile, 0.1% TFA solution and discard the flowthrough.
9，Elute the labeled glycoprotiens
Analysis of V-tag-Labeled Glycopeptides
U/HPLC analysis
2，Sample injected: 25 µL
3，Injection mode: partial loop, 50 μL loop, syringe solution 80% acetonitrile
4，Eluent A: 50 mMol Ammonium Formate, pH = 4.4
5，Eluent B: Acetonitrile.
6，Temperature: 60 °C
7，Detection: Fluorescence, λex = 250 nm, λem = 360 nm