Procainamide Labeling Kit-Sodium cyanoborohydride-96T
Product description
Application: For labeling free glycans with procainamide (PROC).
Description: This kit contains reagents for conjugating dyes to the free reducing ends of glycans via reductive amination reactions.
Dye Trait: Massless Dye = 235.33.
Fluorescence, ex = 310 nm, em = 370 nm.
Number of samples 96 individual assay samples per set of labeling reagents
Sample amounts per sample ranged from 25 pmol to 25 nmol glycans.
Appropriate samples can label any purified glycan with free reducing ends.
No detectable (< 2 mol %) loss of structural integrity sialic acid, fucose, sulfate or phosphate.
Label selectivity is essentially stoichiometric labeling.
Storage: Store at room temperature away from light. Keep away from heat, light sources and moisture. The reagents provided are stable for at least two years.
Shipping: This product can be shipped at ambient temperature.
Handling: Make sure any glass, plastic or solvent used is free of glycosidases and environmental carbohydrates. Use powder-free gloves during all sample handling procedures and avoid contamination with environmental carbohydrates. All steps involving labeling reagents must be performed in a dry environment using dry glassware and plasticware. Once individual reagent vials are opened, their contents should be used immediately, excess quantities then discarded in accordance with local safety regulations.
Safety: For Research Use Only. Not for human or medicinal use
Kit Contents
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Additional reagents and equipment required
pure water
Heat block, oven or similar dry heater (no water bath) set to 65°C
Centrifugal evaporators (eg Savant, Heto or similar)
Reaction vials (eg polypropylene microcentrifuge vials)
NOTE: Additional consumables are required if optional post-marking sample cleanup is performed (see sample cleanup section)
Timeline of labels
The labeling process takes 2.5 hours, while the actual labeling incubation takes only 1 hour.
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Labeling method
1 Purified glycans
Before labeling glycans, it is best to remove non-carbohydrate contaminants such as protein/peptide material, salts and detergents from the sample. This can be achieved using EB10 filter cartridges or protein-bound membranes. Purification of O-glycans can be achieved using CEX cartridges.
2 Transfer the sample to a reaction vial or PCR plate
The kit is designed to label up to 25 nmol of glycans per reaction. Using a single pure glycan, as low as 5 pmoles per reaction can be labeled and detected in subsequent HPLC and MS analysis.
Suitable reaction vials include small polypropylene microcentrifuge vials, PCR work vials, or 96 PCR plates for higher throughput sample analysis.
3 dry samples
Drying of samples should be performed using a centrifugal evaporator. If this is not possible, freeze-drying (lyophilization) can be used sparingly (especially to ensure that the sample dries to a small, dense mass at the bottom of the vial). Do not expose samples to high temperature (>28°C) or extreme pH conditions, as these conditions may lead to acid-catalyzed loss of sialic acid (high temperature, low pH) or glycan reducing end epimerization (at high pH conditions) Down).
4 Prepare the dye solution
Add 1.2 mL of kit component LT‑ACETIC‑DMSO 96 (30% acetic acid in DMSO) to the procainamide dye bottle, cap and shake and vortex until the dye is dissolved.
Add 1.2 mL of water to the dissolved dye solution, cap and shake and vortex.
Transfer 2.4 mL of the dissolved dye solution to a bottle of reducing agent, cap and shake and vortex until the reducing agent is completely dissolved.
5 Add labeling reagent to the sample
Add 20 µL of labeling reagent to each glycan sample, cap the vial or PCR plate, mix thoroughly and centrifuge briefly (5‑10 seconds) to ensure the labeling solution is at the bottom of the vial.
6 Incubation
Place the reaction vial in an oven or heat block set to 65°C and incubate for 1 hour. We recommend using an oven for the incubation step. The oven provides a full range of heat to the reaction vial.
The sample must be completely dissolved in the labeling solution for effective labeling. Encouraging completion of lysis After 15 minutes of initial incubation, samples can be vortexed before continuing.
7 Cooling and centrifugation
After the incubation period, remove the samples from the oven or heat block, allow them to cool to room temperature, and briefly centrifuge the reaction vial to ensure that the sample is in the bottom of the vial and not in the lid.
Post-labeling sample cleanup
For most applications, we recommend that you perform post-labeling sample cleanup to remove nonglycan materials such as excess dyes and other labeling reagents prior to analysis by HPLC, MS, or LC‑MS.
The benefits of post-label cleaning
Removes free dyes and chemicals that can interfere with HPLC and LC‑MS sample analysis.
Extend the life of HPLC columns.
Smaller glycans such as O-link near the start of the HPLC gradient will be detected without interference from free dyes.
A full range of HPLC columns can be used.
Sample capacity issues with HPLC columns are unlikely to occur.
Post-label sample cleanup of procainamide (PROC)-labeled N-glycans can be achieved using S-columns or the Procainamide Clean-up Plate if performing higher throughput sample analysis.
Post-labeling sample cleanup of PROC-labeled O-glycans can be performed using S-cartridges.
Analysis of procainamide-labeled glycans
Procainamide-labeled glycans can be studied by a number of different analytical methods, including (U)HPLC, ESI mass spectrometry, and LC‑ESI‑MS.
Example UHPLC Conditions – Conditions vary by instrument. Check if these conditions are suitable for your device.
Samples are prepared in a solvent equivalent to the starting gradient, eg 76% acetonitrile.
Column: Waters BEH Glycan column 15 cm x 2.1 mm.
Column temperature: 40°C
Fluorescence detector settings: Excitation wavelength: 310 nm, Emission wavelength: 370 nm.
Solvent A: 50 mM ammonium formate buffer pH 4.4 (Ludger product: LS‑N‑BUFFX40)
Solvent B: Acetonitrile
Long UHPLC gradients for samples with unknown glycan profiles and glycans may be large and/or highly sialylated.
The use of this UHPLC gradient was demonstrated to separate N-glycans released from human IgG and erythropoietin, followed by procainamide labeling and cleanup. Once the range of glycans present in the sample is determined, the UHPLC gradient can be optimized and shortened significantly (to 15 minutes on some UHPLC instruments).
LC-MS analysis of procainamide-labeled N-glycans allows the user to obtain fluorescence chromatograms and ESI-MS spectra of isolated N-glycans, which can be combined for a more complete understanding of N-glycans The chain structure is present. MS data can be used to assign an m/z mass to each peak in a fluorescence chromatogram, giving each peak possible structural identity in terms of monosaccharide composition. Through ESI‑MS analysis, a single peak in the fluorescence chromatogram can be identified with multiple glycan structures.
Purified procainamide-labeled human IgG glycans
Purified procainamide-labeled erythropoietin glycans
Reductive amination reaction
1. Schiff base formation
This requires glycans with free reducing ends that balance between closed (cyclic) and open (acyclic) forms. The primary amino group of the dye conducts a nucleophilic attack on the carbonyl carbon of the acyclic reducing end residue to form a partially stable Schiff base.
2. Reduce Schiff base.
The Schiff base imine group is chemically reduced to produce stable labeled glycans.
Glycans are labeled with procainamide (PROC) by reductive amination.
References
1.Kozak RP, Tortosa CB, Fernandez DL, Spencer DI. Comparison of procainamide and 2-aminobenzamide labeling for the analysis and identification of glycans by fluorescence detection by liquid chromatography coupled to electrospray ionization mass spectrometry. Anal biochemistry. 2015;486:38‑40. doi: 10.1016/j.ab.2015.06.006.
2. Klapoetke S, Zhang J, Becht S, Gu X, Ding X. Evaluation of a new method for the analysis and identification of procainamide-tagged N-linked glycans by HPLC with fluorescence and mass spectrometry detection. J Pharm Biomedical Anal. 2010;53(3):315‑24. doi: 10.1016/j.jpba.2010.03.045.
3.Pabst M, Kolarich D, Pöltl G, Dalik T, Lubec G, Hofinger A, Altmann F. Comparison of fluorescent labeling of oligosaccharides and introduction of new post-labeling purification methods. Anal biochemistry. 2009;384(2):263‑73. doi: 10.1016/j.ab.2008.09.041.
4. Liu R, Giddens J, McClung CM, Magnelli PE, Wang LX, Guthrie EP. Evaluation of glycoengineered monoclonal antibodies by LC‑MS analysis combined with multiple enzymatic digestions. Monoclonal antibodies. 2016;8(2):340‑6. doi: 10.1080/19420862.2015.1113361.