Monosaccharide Release and Labeling Kit-96T

Product description
Application：For release of neutral and amino monosaccharides from glycoprotein therapeutics and pre-released glycans, and subsequent labeling with 2-aminobenzoic acid (2-AA).

Description：The kit contains reagents for the release of monosaccharides from glycoprotein biopharmaceuticals and standards. Released monosaccharides have a free reducing terminus to allow fluorescent tagging by reductive amination. There are two hydrolysis acids provided. 2 molar trifluoroacetic acid (2M TFA) and 6 molar hydrochloric acid (6M HCl). Typically, for a pilot study, we recommend using both these acids on separate replicates of your samples as we have found that 2M TFA is good for releasing neutral monosaccharides but is less effective with the N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) monosaccharide release for which we recommend using 6M HCl. After a pilot study it may be determined that the use of just a 6M HCl hydrolysis step may be sufficient for quantitative analysis of your glycoprotein. Hydrochloric acid as well as providing more effective release of GlcNAc may also aid core fucose release and core mannose release; however these latter monosaccharides are subject to degradation by HCl so care should be taken to perform hydrolysis in a consistent manner and we recommend a simultaneous hydrolysis of the monosaccharide standards for calibration purposes.

Number of Samples：The kit contains reagents and materials for up to 96 glycoprotein samples analysed in parallel or two sets of 48 samples. 

Amount of Sample：Typically, around 50 µg of glycoprotein per sample dependent on levels of glycans present which should be determined in a pilot study.

Suitable Samples：Biopharmaceutical glycoproteins.

Storage：Store the whole kit at 4°C in the dark. Once monosaccharide standards are dissolved in solvent we recommend storing them at -20°C. Protect from sources of heat, light, and 

moisture. Use kit within 6 months of purchase.

Shipping ：The product should be shipped at ambient temperature, but can be stored at 4°C for up to 6 months.

Kit Contents

The kit contains the following materials and reagents:

Item	Quantity
Trifluoroacetic acid	2
Hydrochloric acid	2
Sodium acetate	2
Labeling solvent	2
2-aminobenzoic acid	2
Sodium cyanoborohydride	2
10 nmols each of glucosamine, galactosamine,galactose,glucose (dextrose) mannose and fucose	4
100 nmol of xylose	2

Additional Reagents and Equipment Required

• Pure water: resistivity above 18 MΩ-cm, particle free (>0.22 μm), TOC <10 ppb

• Vials - Screw cap polypropylene vials for hydrolysis. 0.5 mL volume size is ideal. Vials lids should seal tightly and have O-rings to prevent escape of acid vapour. We do not recommend the use of snap cap type vials such as used for PCR unless they are designed to fit securely without clamping.

• Oven for incubation stages (recommended) or heating block. Note that the use of a heating block often results in some sample solvent evaporation into the vial lid which may reduce the effectiveness of sample hydrolysis or labeling.

• Centrifugal evaporator

• Vortexer and sonicator

• Pipettes

• For the HPLC solvent: Butylamine, >99.5%

                                      Tetrahydrofuran, anhydrous >99.9%, BHT inhibitor

                                      Orthophosphoric acid about 85% concentration

                                       Acetonitrile

• Optional - depending on your sample. High levels of salts may inhibit fluorophore labeling. The xylose 

  internal control can be used as a guide as to whether high salt labeling inhibition is occurring

Time Line for Procedure

The labeling procedure typically takes 14 hours

Procedure	Approx Time
Dispense and dry samples(samples can be stored at -20 °C until required)	4h
Hydrolyse samples	3h
Cool and dry samples(samples can be stored at -20 °C until required)	4h
Add labeling reagents	1h
Chemically label	1h
Dilute samples	1h

Instructions for Use
Dispense and dry samples

1. Dispense 50 µg of each glycoprotein sample into a 0.5 mL screw cap polypropylene vial in triplicate.

Two samples are required for each analysis – one for 2M TFA hydrolysis and one for 6M HCl hydrolysis. Ideally each solution should be no greater than 200 µL in volume to prevent sample loss out of the vial during the centrifugal drying process. The use of 0.5 mL screw cap vials with O-rings in the lids is recommended (snap cap lid type vials may fail during the hydrolysis step).

2 .Sample Blank - Dispense equivalent volume of sample buffer into a 0.5 mL screw cap polypropylene vial in triplicate. 

3 .Dry samples by centrifugal evaporation   Do not apply heat. Samples should be dry within three hours dependent on vacuum efficiency.

Hydrolyse samples

4. To dried sample add 200 µL For neutral monosaccharide analysis (Gal, Man, Glc, Fuc, Xyl)

Vortex for 10 seconds and tighten cap to ensure no evaporation from tube during heating step. 

At this stage you should also add acid to the dry 10 nmol monosaccharide standards (CM-Mono-Mix-10).

5. To dried sample add 200 µL For N-acetylgalactosamine and Nacetylglucosamine analysis (GalNAc, GlcNAc)

6M HCl is more effective at releasing GalNAc and GlcNAc (and possibly core fucose and mannose)monosaccharides than 2M TFA but will partially destroy the neutral monosaccharides. 

6. Place vials in oven 

Heat samples at 100° C for 3 hours in an oven. The samples should be re-mixed after 30 min to ensure dissolution of the glycoprotein in the acid. If a hot plate is used instead of an oven smaller vials will need to be used or a larger volume of acid to prevent all the acid from evaporating and condensing on the sample vial lid and not sufficiently hydrolysing the sample.

Please note that there are many different hydrolysis conditions reported in the scientific literature and we recommend that you optimise these conditions with your own glycoproteins.

Cool and then dry samples

7. Remove vials from oven and allow them to cool. Centrifuge to ensure all sample is in the vial and not the cap.

8. Add xylose internal standard to each sample.

We recommend the use of the xylose monosaccharide as an internal standard. We provide a 100 nmol amount of xylose for this purpose. Dissolve the xylose in a set amount of water (e.g. 200 µl), mix thoroughly to ensure complete dissolution and then add a set amount to each test sample (e.g. 10 nmol or 20 µl dissolved sample). Often the use of xylose is more effective in a full study after a preliminary investigation has already been performed on the samples and any presence of xylose and the absolute levels of monosaccharides in the test samples have been determined. Xylose is not recommended as a standard for proteins expressed by plant-cell based expression systems.

9. Loosen vial caps and evaporate the acid in a centrifugal evaporator

This stage should take up to 4 hours. After 2 hours of drying check that vial cap O-rings are not sealing the tube preventing sample drying. Many samples will dry to a dark brown or black spot.

Chemically label samples
10.Preheat oven/hot plate to 80 °C

Note that we recommend the use of an oven as this prevents any solution condensing in the sample cap.

11.Incubate samples for 45 minutes at 80° C，Ensure that the vial lids are tight.

Dilute samples

12. Remove samples from incubator and allow to cool

Cool the samples to room temperature and then briefly spin in a centrifuge to remove condensate from vial cap.

13. Dilute samples Once cooled and centrifuged the labeled samples are ready for analysis concentrated to be injected onto the HPLC column. Samples are diluted into the butylamine/ortho-phosphoric acid/tetrahydrofuran (BPT) HPLC running solvent. For the monosaccharide standards we recommend diluting the samples 100 fold, but exact dilutions are dependent on the sensitivity of the fluorescence detector and the glycan content of the glycoprotein. See the calibration curve section of this document. As a guide, antibody samples should be diluted 20 to 50 fold as there is often only two glycosylation sites per molecule. Bovine fetuin glycoprotein standard is usually run at 100 fold dilution, where fetuin is approximately 20% w/w glycan. After samples dilution we recommend sonicating the samples for 30 minutes to remove any gas produced at the dilution stage. Vials can remain capped during sonication. If a sonicator is not available samples can be left for approximately 12 hours after dilution and then lightly mix to remove bubbles. No further gas release will occur after 12 hours.

HPLC analysis of samples

Solvents

The glycan analysis gradients in this guide are based on the following solvents:

Solvent A : purified water based solvent of 0.2 % butylamine (2 mL per litre), 0.5 % phosphoric acid (5 mL per litre), 1 % tetrahydrofuran (10 mL per litre) (henceforth called BPT solvent). Solvent B : 50 % acetonitrile : 50 % solvent A We have also tested acetonitrile free solvent systems. This requires a slightly different gradient. Please enquire for further details. Column should be stored in 30% solvent B long term.

We have also tested acetonitrile free solvent systems. This requires a slightly different gradient. Please enquire for further details. Column should be stored in 30% solvent B long term.

Gradient 
Column temperature: 30 °C Fluorescence detector settings : Excitation wavelength: 360 nm, Emission wavelength: 425 nm

Time(min)	%B	Flow Rate(ml/min)
0.00	7	0.8
7.00	7	0.8
25	17	0.8
26	100	0.8
36	100	0.8
37	7	0.8
45	7	0.8

Calibration Curve

We recommend a five point calibration curve to be used for the monosaccharide standards. If you have used both HCl and TFA hydrolysis conditions you will need to perform a calibration curve for a set of monosaccharide standards hydrolyzed with each acid. Dilute the hydrolyzed monosaccharide mixtures into BPT solvent. We recommend the following dilutions, but these may vary dependent on the glycosylation levels in your sample. Always run a standard curve that allows you to place your sample results within the curve. Pipetting larger volumes than 5 µl is generally more accurate.

1in 100 (25 µl hydrolyzed monomix standard + 2475 µl BPT)

1in 125 (20 µl hydrolyzed monomix standard + 2480 µl BPT)

1in 167 (15 µl hydrolyzed monomix standard + 2485 µl BPT)

1in 250 (20 µl hydrolyzed monomix standard + 4980 µl BPT)

1in 500 (10 µl hydrolyzed monomix standard + 4990 µl BPT)

Sonicate samples to remove gas bubbles. The vial lid does not need to be loosened for this stage.

Data Interpretation

HPLC analysis of 2-AA monosaccharides is a robust method for determining monosaccharide levels in your glycoprotein sample. There are some important issues that need to be understood to enable effective data interpretation.

1. Each monosaccharide has a different fluorescence yield per nmol. A standard curve of monosaccharide concentration versus fluorescence needs to be determined.

2. Free dye is not removed from the samples after labeling to ensure no monosaccharides are lost in a dye clean-up stage. Free dye elutes as a large off-scale peak at about 15 minutes (Figure 1), and also several large peaks after 28 minutes.

3. N-acetylglucosamine and N-acetylgalactosamine are both de-N-acetylated during the acid hydrolysis step of this process to produce glucosamine and galactosamine respectively.

4. Glucosamine is subject to an approximate 4 % epimerisation resulting in mannosamine (Figure 2). Mannosamine elutes immediately before the galactosamine peak, with no baseline resolution.

5. Galactosamine also produces an epimer peak (Figure 1 – small peak immediately after GalN), typically smaller than 2 %. However this epimer peak often elutes with the free dye peak.

6. The use of xylose as an internal standard (Figure 3) allows samples to be compared directly. This allows compensation for any pipetting/sample preparation errors that may have occurred during sample processing.

7. Sample stability – we recommend keeping labeled samples in the freezer at -20 °C if you intend to store them for longer than one week.

Figure 1: fluorescence chromatogram of monosaccharides glucosamine (GlcN), galactosamine (GalN), galactose (Gal), mannose (Man) and fucose (Fuc). Glucose elutes immediately after the Man peak. Xylose elutes before the Fuc peak.

Figure 2: fluorescence chromatogram of monosaccharide glucosamine (GlcN). This chromatogram shows the epimer peak from GlcN. 

Figure 3: fluorescence chromatogram of monosaccharide internal reference xylose (Xyl).

Troubleshooting Guide

The following is a guide to the most likely problems associated with monosaccharide analysis, possible causes, and solutions.

Low Yield or High Sample to Sample Variability

The sample was incompletely solubilized.

The hydrolysed samples can be difficult to dissolve in 1 % sodium acetate prior to labeling. In the case of a black/brown hydrolysed sample it is clear when the sample has not dissolved, however, not all sample pellets are readily visible. To ensure good solubility of the sample, shake/sonicate the sample vials rather than just vortexing them. Once dissolved, centrifuge the sample briefly.

Variability in Pipetting

Ensure that pipettes are calibrated and that the pipette tips used are maximum recovery type.

Diluted samples not sufficiently degassed. Gas production/displacement occurs in the samples shortly after dilution. We recommend running the samples on the HPLC after this process of degassing has finished (up to 12 hours after dilution if left to stand in a refrigerator, or after 30 minutes in a sonication bath) otherwise air bubbles can be injected onto the HPLC column causing large sample to sample variation. The gas released is low enough that sample vial caps do not need to be loosened.

Precipitation in sample vials

Large amount of dye/reductant excess

In order to get good labelling efficiency a large molar excess of dye and reductant to free monosaccharide can be used. This can sometimes result in a cloudy precipitate forming in samples. Reducing the concentration of dye and reductant in the labelling reaction may help to prevent the precipitation occurring.

Old solvents Ensure that HPLC solvents are made up fresh for each chromatography run. Inconsistent solvent preparation Ensure that solvent components are dispensed accurately. Sometimes pipette tips drip because they are not compatible with the solvent being dispensed. Weighing of solvent components is often used as an alternative to the use of volumes when dispensing. 

Old solvents

Ensure that HPLC solvents are made up fresh for each chromatography run.

Inconsistent solvent preparation

Ensure that solvent components are dispensed accurately. Sometimes pipette tips drip because they are not compatible with the solvent being dispensed. Weighing of solvent components is often used as an alternative to the use of volumes when dispensing.