[ CARE AND USE MANUAL ]
GlycoWorks High-throughput Sample Preparation Kit
CONTENTS
I. INTRODUCTION
II. STORAGE AND DISPOSAL OF PLATES
III. ABBREVIATIONS
IV. USING THE GLYCOWORKS HIGH-THROUGHPUT KIT
V. PREPARING LABELED GLYCANS FOR HILIC-FLR WITH
AN ACQUITY UPLC BEH GLYCAN COLUMN
VI. REFERENCES
VII. ORDERING INFORMATION
I. INTRODUCTION
This document provides information regarding the general care and
use of the Waters GlycoWorks™ High-throughput Sample Preparation
Kit for release of N-glycans. T his protocol can be used as a guideline
and is validated using IgG but can also be used and optimized for
other N-linked glycoproteins. This sample preparation offers an
inclusive protocol that allows the user to source many of the critical
components needed to perform the preparation from one vendor.
General Guideline of Sample Preparation from Glycoprotein to
enrich FLR Labeled Glycans Using Reductive Amination Reaction.
Glycoproteins
Denaturation and Deglycosylation
1
2
3
4
RapiGest™ (if necessary)
DTT Reduction (if necessary),
IAM Alkylation (if necessary)
Deglycosylation by PNGase F
Extraction of Released Glycans
GlycoWorks HILIC µElution Plate
FLR Labeling Reaction of Glycans
Excess Labeling Reagent Removal
GlycoWorks HILIC µElution Plate
FLR Labeled Glycans
MALDI MS
LC-FLR
LC-FLR-MS
The 96-well plate is good for 48 analyses.
For expected results using the protocol with the control standard,
please see Reference 1.
The guide provides the following:
■
An instruction template for new users that highlights the use of a
control standard (included in the kit) for first time validation as
well as subsequent troubleshooting
■
Protocol for LC/FLR and LC/FLR/MS
■
Tips and tricks throughout each section to help aid in the success
■
An online training video is also available (see Reference 3)
GlycoWorks High-throughput Sample Preparation Kit
If using MALDI please refer to Product Solution, Literature Code 720004660EN
Figure 1: Sample Preparation from Glycoprotein to Labeled Glycans
1
[ CARE AND USE MANUAL ]
Table 1: Kit Components
Kit Component Purpose
GlycoWorks HILIC
µElution Plate
GlycoWorks Glycoprotein
Control Standard
RapiGest SF An enzyme friendly detergent used
Manifold Waste Tray For collection of non-releasing glycans
GlycoWorks Reagent Kit Contains ready to use reagents for both
Extract and clean up of free or FLR
labeled glycans
■
Capture of glycans
■
Removing contaminants like salt and
detergents from hydrophilic analytes,
such as carbohydrates, prior to MS
■
Removing excess
labeling reagent
■
The 5 mg sorbent was test to have
maximum binding capacity for 200 µg
glycans
Unlabeled IgG (100 µg/lyophilized) to
use as a control for process validation and
troubleshooting
to denature the glycoproteins prior to
deglycosylation reaction. For stability
purposes, there is enough RapiGest included
for initial use of the kit with 5 samples, more
may need to be ordered for processing all 46
samples.
steps. This is disposable but can be
cleaned and used several times before
ac quiring a new one as long as it is rinsed
out.
denaturation, reduction, and alkylation as
well as raw materials for FLR tagging.
II. STORAGE AND DISPOSAL OF PLATES
Plates stored in their original sealed pouch remain stable for long
periods. To store unused plates in open pouches, squeeze the air out
of the pouch, fold over the open end of the pouch twice, seal with
tape, and store in a desiccator.
Note: Dispose of used plates safely in accordance with applicable
government or local regulations
III. ABBREVIATIONS
IV. USING THE GLYCOWORKS HIGH-THROUGHPUT KIT
Below, are general guidelines for utilizing the GlycoWorks High-
throughput Kit for complete extraction, purification and concentration
of N-linked glycans validated using IgG. Also included is a check list of
accessory materials that will be needed to complete the protocol from
start to finish as well as tips and tricks throughout the instructions.
Table 2: Checklist of Additional Materials Needed Before You Begin
Material
Description
Ammonium
bicarbonate
Ammonium acetate
Formic acid (to make
1% [v/v] formic acid,
prepared in 50%
acetonitrile)
15,000 units
(500,000 units/mL)
PNGase F*
Acetonitrile
25 mM ammonium
bicarbonate in water,
pH 8–9
100 mM ammonium
acetate in 5% [v/v]
acetonitrile, pH 7
85% [v/v] acetonitrile See Above Yes No
96-well collection plate
2-AB** Sigma P/N A89804 Yes No
µElution plate
manifold
SPE vacuum pump
Positive pressure
manifold
Centrifugal vacuum
evaporator
Heat block N/A Ye s No
Recommended
Suppliers
Sigma Aldrich
Cat No. O9830
Sigma Aldrich
Cat No. 73594 FLUKA
Sigma Aldrich
Cat No. F0507
New England Biolabs
Cat. No. P0704S
Sigma Aldrich
Cat No. 360457
See Above Yes No
See Above Yes No
Waters P/N
186 002481
Waters P/N
186 001831
115, 60 Hz:
Waters P/N 176002986
Waters P/N
186 006961
N/A Ye s No
Check List:
Circle
Yes No
Yes No
Yes No
Yes No
Yes No
Yes No
Yes No
Yes No
Yes No
DMSO Dimethylsulfoxide IAM Iodoacetamide
DTT Dithiothreitol 2AB 2-aminobenzamide
GlycoWorks High-throughput Sample Preparation Kit 2
* PNGase F comparison table: https://www.neb.com/tools-and-resources/
usage-guidelines/glycobiology-unit-conversion-chart
**Inclusive Kits are also offered by ProZyme, Ludger, and Sigma covered
under US Patent No. 5,747,347 and its foreign equivalents.
[ CARE AND USE MANUAL ]
Step 1. Denaturation and Enzymatic Deglycosylation
RapiGest, DTT, IAM are included in the GlycoWorks Reagent Kit box labeled
“Enzymatic Deglycosylation Reagents” for your convenience in following
this protocol:
1) Prepare RapiGest solution by adding 500 μL of 25 mM ammonium
bicarbonate to 1 mg RapiGest vial.
2) For the GlycoWorks Control Standard, add 100 μL of 25 mM
ammonium bicarbonate to vial.
3) Add 90 µL of the 0.2% RapiGest solution to the glycoprotein
solution in a sterile 1.5 mL centrifuge tube.
Note: For use with other samples, it is recommended that the protein
concentration be 1–5 mg/ml.
3) Prepare 0.5 M DTT by adding 500 µL of 25 mM ammonium bicarbon-
ate to the tube. Vortex to mix into solution.
■
Add 2 μL of 0.5 M DTT and incubate sample(s) at 37 °C for
30 minutes. Cool to room temperature.
4) Prepare 0.5 M IAM by adding 500 µL of 25 mM ammonium
bicarbonate to the tube. Vortex to mix into solution.
■
Iodoacetamide is light sensitive. Cover the tube in foil and
place it in a dark drawer when not in use. T his solution should
be made fresh daily.
■
Add 4 μL of 0.5 M IAM and incubate at room temperature in
the dark for 30 minutes.
5) Add 2.5 mU (1 μL of 2.5 mU μL-1 stock) of PNGase F to each
sample and incubate at 37 °C overnight. If a more concentrated
enzyme stock is purchased, dilute with included buffer first to get
to a 2.5 mU/µl concentration.
Note: If digesting glycoprotein samples greater than 1 mg/mL, it is
recommended to use more PNGase F.
Step 1 Tips and Tricks
■
The minimum amount of glycoprotein advised is 10 μg
(100 μL of 0.1 mg/mL).
Step 2a. Extracting the Released Glycans
Note: There may be variation in flow rates from well to well when using
this plate with the vacuum manifold. General guidelines are provided to
help minimize this. However, settings will depend on the specific vacuum
and will need to be optimized. If well to well reproducibility is critical,
using a positive pressure manifold produces consistent reproducibility.
1) Condition the GlycoWorks HILIC μElution plate by adding 200 μL of
Milli-Q® water to each well and aspirate using the vacuum manifold
or positive pressure manifold.
2) Add 200 μL of 85% acetonitrile to each well and aspirate using the
vacuum manifold or positive pressure manifold.
3) Take 50 μL of the PNGase F-digested protein mix and add this to
350 μL of acetonitrile.
Note: If precipitation occurs, do not centrifuge! Centrifugation causes
reduced glycan recovery.
4) Load the sample onto the resin bed using a very low vacuum setting
(1–2 in Hg) or positive pressure manifold.
5) Wash each well 3 times with 200 µL of 85% acetonitrile, pooling
the filtrate after each wash into the waste tray for disposal.
6) Remove the waste tray and replace with a 96-well collection plate.
7) Elute the glycans with 50 μL of 100 mM ammonium acetate in 5%
acetonitrile.
Note: The concentration of acetonitrile and ammonium acetate in the
elution buffer may require optimization for some glycan samples.
8) Repeat the elution two more times.
9) Place the collection plate and dry released glycans by vacuum
evaporation to a concentrate of ≤ 1 µL in volume.
Note: If a rotor is not available for the vacuum centrifugal evaporator,
transfer each collected elution into a sterile 1.5 (or 0.5) mL Eppendorf
and dry down by vacuum evaporation.
■
Avoid temperature extremes with glycan samples.
■
Exposure to high pH may result in epimerization of
reducing sugars.
GlycoWorks High-throughput Sample Preparation Kit 3
[ CARE AND USE MANUAL ]
General Guidelines for Vacuum Settings
■
For loading, washing, and elution a setting of 1–2 in Hg is
suggested.
■
For conditioning steps higher vacuum can be employed.
■
If no flow is observed, there may be an air bubble. Employ
high vacuum to initiate flow then return to an appropriate
vacuum setting.
General Guidelines for Positive Pressure Manifold:
■
For loading, washing, and elution, approximately 3 psi is
appropriate.
■
For conditioning, 10–20 psi can be used.
Step 2b. Formic Acid Treatment of Released Glycans
1) Prepare 1% formic acid by adding 10 μL formic acid to 990 μL of
50% acetonitrile.
2) Add 50 μL of 1% formic acid solution to each glycan sample.
Step 3. Labeling of Glycans
Reference: Legal Information
Labeling of glycans with 2-AB is covered under US Patent No. 5,747,347
and its foreign equivalents.
Below is only an example of a sample procedure that one can follow
for labeling. Acetic Acid, DMSO, and Sodium cyanoborahydride are
included in the GlycoWorks Reagent Kit box labeled “Additional
Reagents” for your convenience in following this protocol with the
control standard.
Note: Prepare labeling solution immediately prior to labeling.
1) Add 300 μL of acetic acid vial to 700 μL of DMSO vial in the kit.
2) Weigh out 10 mg of 2AB.
3) Add 800 μL of the acetic acid/DMSO mixture to the entire vial of
chosen label.
4) Mix until dissolved (may require vortexing).
5) Add the entire contents of the labeled mixture to the vial of sodium
cyanoborohydride in the kit and mix until completely dissolved.
3) Incubate for 40 minutes at room temperature.
4) Dry glycans using vacuum evaporation bringing them to
complete dryness.
Step 2 Tips and Tricks
■
The concentration of ammonium acetate and acetonitrile in
the elution buffers of Steps 2a and 4 may require optimization for
some glycans.
■
The reason for using low concentrations of formic acid is to
convert all glycans to free reducing glycans, hence improving
the overall yield of the FLR-labeling via reductive amination.
■
Drying down the sample does cause some sample loss but it
essential to have the sample completely dry before proceeding
to Step 3.
6) Add 10 μL of the labeling solution to each dried glycan sample.
Ensure glycans are fully reconstituted in the 2AB label.
7) Incubate glycan samples for a minimum of 3 h at 65 °C in a heating
block (if samples are in eppendorfs), dry oven, or sand tray.
8) Following incubation, briefly spin the vials to recollect each sample
at the bottom of the vial if samples are in Eppendorfs.
9) Allow samples to cool to room temperature.
Step 3 Tips and Tricks
■
Protect the final preparation of labeling reagent from light.
■
The sodium cyanoborohydride once it is solubilized, should be
used within an hour.
■
Left over reagent which contains sodium cyanoborohydride has
to be disposed separately in a clearly marked bottle due to its
toxicity (see MSDS).
■
To ensure high yield of labeled glycans, it may be necessary,
for some samples, to increase the concentrations of the
reducing and labeling reagents.
GlycoWorks High-throughput Sample Preparation Kit 4
[ CARE AND USE MANUAL ]
Step 4. Removal of Excess Label
1) Condition a NEW well of the Glycoworks μElution plate by add-
ing 200 μL of Milli-Q water to each well and aspirate using the
vacuum manifold or positive pressure manifold.
2) Add 200 μL of 85% acetonitrile to each well and aspirate using
the vacuum manifold or positive pressure manifold.
3) Add 100 μL of acetonitrile to the 10 μL of 2AB labeled glycan
sample and load into appropriate well on GlycoWorks HILIC
μElution plate using low vacuum setting (1-2 in Hg) or positive
pressure manifold.
4) Wash each well with 200 μL of 85% acetonitrile. Remove
filtrate to waste.
5) Repeat the washing step two more times.
6) Replace the waste tray with a 96-well collection plate.
7) Elute glycans with 50 μL of 100 mM ammonium acetate in 5%
acetonitrile.
Note: The concentration of ammonium acetate and acetonitrile in the
elution buffer may require optimization for some glycan samples.
V. PREPARING LABELED GLYCANS FOR HILIC-FLR WITH
AN ACQUITY UPLC BEH GLYCAN COLUMN
The following steps are suggested for preparing the labeled glycans for
HILIC-FLR with an ACQUITY UPLC BEH GLYCAN column when using this
kit with the Waters Total Glycan Application Solution.
1. Reconstitute the labeled glycans in 50 µL of Milli-Q water by
gently aspirating, avoiding vortexing and sonication.
2. Add 75 µL of ACN to the reconstituted glycans immediately prior
to and in preparation for analysis by HILIC-FLR.
3. Inject 4 µL of the labeled glycan containing 60% ACN solution
onto the column.
Note: Preparations of non-IgG glycoproteins or glycoprotein samples with
concentrations other than 1 mg/mL may need to be reconstituted with
different volumes to achieve desired results. Similarly, more concentrated
samples can be obtained by reconstituting with up to 10 times lower volumes.
The optimum injection volume for this application is ≤4 µL.
VI. REFERENCES
8) Repeat elution two more times.
9) Place 96-well collection plate in a centrifugal vacuum evaporator
and dry the glycans to a concentrate of ≤ 1 µL in volume.
Note: If a rotor is not available for plates, transfer each eluent to a sterile
1.5 (or 0.5) mL eppendorf and place in the vacuum evaporator. This may
contribute to a small sample loss due to extra transfer.
10) Glycans can be stored in Milli-Q water at -20 °C until required.
Step 4 Tips and Tricks
■
Extended periods of time between incubation and analysis
may result in desialylation of labeled glycans and consequently
should be avoided.
1. Lauber MA, Koza S, Fountain K J. Optimization of HILIC SP E for the Quantitative and Robust Recovery of N-Linked Glycans. Waters Application Note
720004710EN. 2013 June.
2. Lauber MA, Koza S, Fountain KJ.Single-Use and High-Throughput HILIC SPE
Device Formats and an IgG Control Standard for Facilitating N-Glycan Analy-
ses Waters Technology Brief 720004711EN. 2013 June.
3. Training Video: http://www.waters.com/waters/en_US/Glycan-Standards/nav.
htm?cid=134640534
GlycoWorks High-throughput Sample Preparation Kit 5
[ CARE AND USE MANUAL ]
VI. ORDERING INFORMATION
Glycan Sample Preparation Kit and Standards
Description Part No.
GlycoWorks High-throughput Prep Kit
GlycoWorks HILIC µElution Plate, 96-well
RapiGest SF 1 mg Vial
GlycoWorks Control Standard, 100 µg Vial
GlycoWorks Reagent Kit
Manifold Waste Tray
GlycoWorks Single Use Prep Kit
GlycoWorks HILIC 1 cc Cartridge (20/pk)
RapiGest SF 1 mg Vial
GlycoWorks Control Standard, 100 μg Vial
GlycoWorks Reagent Kit
Glycan Performance Test Standard
The Glycan Performance Test Standard is a 2-AB labeled
human IgG-like standard that is QC verified to contain
the components needed to benchmark and evaluate
ACQUITY UP LC BEH Glycan, 1.7 µm Columns.
Dextran C alibration Ladder
The 2-AB labeled, Dextran Calibration Ladder is used
to calibrate the HILIC column from retention time to GU
values. This calibration ladder provides good peak shape
and reliable identification from 2 to 30 Glucose Units.
GlycoWorks HILIC 1 cc Flangeless Catridges Pack
This pack of 20 catridges, good for 10 analyses, are
offered for those customers who prefer to use the single
use devices with the positive pressure manifold (PPM).
176003090
186002780
186001860
186007033
186007034
600001282
176 00 3119
186007080
186001860
186007033
186007034
186006349
186006841
186007239
Glycan Analysis System Standards and Mobile Phases
Description Usage Volume Part No.
Alliance with Fluorescence
Qualification Standards Test Kit
Kit contains seven 10 mL vials of varying concentrations of anthracene in
80:20 acetonitrile/water:
(1) 0.5 pg/μL (1) 1.0 pg/μL (2) 5.0 pg/μL
(1) 10.0 pg/μL (1) 2.5 ng/mL (1) 2.5 μg/mL
One blank 10 mL vial of 80:20 acetonitrile/H20
Fluorescence Detector P
Standard Solution
5.0 pg/µL anthracene in
20:80 water/acetonitrile
Fluorescence Detector
Performance P
Standard Solution
0.10 mg/L anthracene in
70:30 acetonitrile/water
Ammonium Formate Concentrate
5000 mM Ammonium Formate in a
Water w/ 3.8% Formic acid matrix.
This allows the end user to dilute the
concentrate 10 mL to 1 L before using to
reach a 50 mM concentration.
C P
C
1 mL
10 m L
10 m L
Calibration P
700002753
700003694
WAT047685
186007081
Performance Check
Additional Consumables
Description Part No.
GlycoWorks HILIC 1 cc
Flangeless Catridges Pack
Dextran Calibration Ladder 200 µg/vial 18 6006841
Glycan Performance Test Standard 228 pmol total /vial 186006349
RapiGest SF 1 mg vial/5 pack 186 001861
96-well Collection Plate 96 wells/50 pack 186 0024 81
Cap Mats for 1 mL Collection Plate 50/pack 186002483
µElution Plate Manifold – 186 001831
SPE Vacuum Pump 115, 60 Hz 176 002 986
Positive P ressure Manif old – 186006961
20/pack 1860 07 239
©2013 Waters Corporation. Waters and T he Science of What's
Possible are registered trademarks of Waters Corporation.
GlycoWorks, and RapiGest are trademark s of Waters Cor poration.
715004079 September 2013 Rev D SC-PDF
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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