deglycosylation conditions and reagents for fast release. In addition, this kit contains a novel rapid label called Rapi Fluor-MS, a reagent purposefully designed to provide both the benefits of sensitive fluorescence detection as well as superb matching signal intensity for mass detection. Finally, sample clean-up prior to LC analyses is accomplished through the use of solid-phase extraction (SPE) with the GlycoWorks HILIC µElution™ Plate. This guide provides highly detailed and informative step-by-step instructions for using this novel protocol. To watch a training video on this protocol, go to waters.com/RapiFluorMS. GlycoWorks Rapi Fluor-MS N -Glycan Kit – 96 Samples CONTENTS I. INTRODUCTION II. STORAGE AND STABILITY III. USING THE GLYCOWORKS RAPI FLUOR-MS N -GLYCAN KIT IV. ORDERING INFORMATION V. GLYCOWORKS RAPI FLUOR-MS QUICK START PROTOCOL VI. REFERENCES AND ADDITIONAL RESOURCES VII. COMMON GLYCANS NAMES, MASSES, AND CHARGE STATES Figure 1. GlycoWorks RapiFluor-MS Start Up Kit. I. INTRODUCTION This document provides information regarding the general care and use of the Waters ® GlycoWorks ® Rapi Fluor-MS ® N-Glycan Kit for the fast enzymatic release and rapid labeling of N-glycans. This protocol is validated using monoclonal antibodies and has also been tested to perform for a wide range of other N-linked glycoproteins. However, the user is advised to confirm enzymatic release for their particular sample. This innovative sample preparation uses optimized Rapi Fluor-MS Reagent Figure 2. From Waters expertise in rapid, fluorescence labeling of amino acids comes the RapiFluor-MS structure. Features of the chemical structure that enable rapid tagging, efficient fluorescence, and enhanced ionization efficiency are highlighted. RapiFluor-MS O NH O N O NH N N O O Rapid Fluorescence MS NHS Carbamate Rapid Tagging Group Quinolinyl Fluorophore Tertiary Amine Charge Tag AccQ·Fluor Rapid Fluorescence [ CARE AND USE MANUAL ]
Transcript
deglycosylation conditions and reagents for fast release. In addition,
this kit contains a novel rapid label called RapiFluor-MS, a reagent
purposefully designed to provide both the benefits of sensitive
fluorescence detection as well as superb matching signal intensity
for mass detection. Finally, sample clean-up prior to LC analyses is
accomplished through the use of solid-phase extraction (SPE) with the
GlycoWorks HILIC µElution™ Plate.
This guide provides highly detailed and informative step-by-step
instructions for using this novel protocol. To watch a training video
on this protocol, go to waters.com/RapiFluorMS.
GlycoWorks RapiFluor-MS N-Glycan Kit – 96 Samples
CONT ENTSI. INTRODUCTION
II. STORAGE AND STABILIT Y
II I . USING THE GLYCOWORKS RAPIFLUOR-MS N -GLYCAN KIT
IV. ORDERING INFORMATION
V. GLYCOWORKS RAPIFLUOR-MS QUICK START P ROTOCOL
VI. REFERENCES AND ADDITIONAL RESOURCES
VII. COMMON GLYCANS NAMES, MASSES, AND CHARGE STATES
Figure 1. GlycoWorks RapiFluor-MS Start Up Kit.
I. INTRODUCTIONThis document provides information regarding the general care and
use of the Waters® GlycoWorks® RapiFluor-MS® N-Glycan Kit for
the fast enzymatic release and rapid labeling of N-glycans. This
protocol is validated using monoclonal antibodies and has also been
tested to perform for a wide range of other N-linked glycoproteins.
However, the user is advised to confirm enzymatic release for their
particular sample. This innovative sample preparation uses optimized
RapiFluor-MS Reagent
Figure 2. From Waters expertise in rapid, fluorescence labeling of amino acids comes the RapiFluor-MS structure. Features of the chemical structure that enable rapid tagging, efficient fluorescence, and enhanced ionization efficiency are highlighted.
Streamlined Protocol for a Simple and Fast Sample Preparation that Minimizes Sample Handling and Loss
Figure 3. Workflow for the rapid preparation of N-glycans using the RapiFluor-MS N-Glycan Kit.
LabeledN-Glycans
LabeledN-Glycans
+ ReagentsReaction
Byproducts
Released N-Glycans + Protein
Glycoprotein
STEP 1: Deglycosylation
STEP 2: Labeling
STEP 3: Clean-up
De
ycans
Labeling
egl cos lationosylationos
entson ucts
5 minutes
10 minutes
O NH
ON
O
NHN
N
O
OOH
N
O
O
+
LC-FLR LC-FLR-MS
Analysis
in as little as 30 minutes
< 15 minutes
2
[ CARE AND USE MANUAL ]
GlycoWorks RapiFluor-MS N-Glycan Kit
Kit Component Purpose Quantity in Kit
GlycoWorks Deglycosylation Module
Intact mAb Mass Check Standard* *Provided in separate box
1 mg of an intact mouse IgG1 monoclonal antibody for use as a control for process validation and troubleshooting.
1 vial
GlycoWorks Rapid Deglycosylation Kit – 4 x 24
GlycoWorks Rapid PNGase F GlycoWorks Rapid PNGase F (Kit Component) = 0.035 mL of PNGase F enzyme for use with rapid deglycosylation procedures. Recombinant source.
4 vials
GlycoWorks Rapid Buffer GlycoWorks Rapid Buffer (0.25 mL of 250 mM HEPES pH 7.9) For use with deglycosylation/labeling.
4 vials
RapiGest™ SF An enzyme-friendly surfactant used to denature the glycoproteins and to facilitate the deglycosylation reaction. Each set of 24 reactions requires one 10 mg vial.
4 vials
GlycoWorks RapiFluor-MS Labeling Module
GlycoWorks RapiFluor-MS Reagent Powder
23 mg/vial of RapiFluor-MS reagent is included for labeling 24 samples. 4 vials
GlycoWorks Reagent Solvent Anhydrous DMF
1 mL ampoule of anhydrous dimethylformamide (DMF) solvent is used for solubilization of the RapiFluor-MS reagent.
4 ampoules
GlycoWorks RFMS Clean-up Module
GlycoWorks HILIC μElution Plate For removing potential interferences like labeling reaction byproducts from the RapiFluor-MS labeled glycans and excess label.
The 5 mg sorbent was tested and found to have maximum binding capacity for 200 μg glycans.
1 plate
GlycoWorks SPE Elution Buffer 5 mL/vial 200 mM ammonium acetate in 5% ACN, eluent that is used for optimum glycan recovery from the HILIC SPE plate.
4 vials
GlycoWorks Sample Diluent 8 mL/vial DMF/ACN Mix-(32/68%) provided to dilute SPE eluate in preparation for HILIC chromatography.
4 vials
GlycoWorks Sample Collection Module
Strips of 8 tubes and caps are included so that an 8-channel pipette can be used to process samples for higher throughput and efficiency.
Deglycosylation Reaction: Caps Optional caps to use during incubation of samples when heating. 15 cap strips of 8
Deglycosylation Reaction: 1 mL Tubes
Recommended for deglycosylation reactions. 15 strips of 8
Labeling Reaction: 600 µL Tubes Recommended for collecting SPE eluate and injecting samples. 15 strips of 8
Labeling Reaction: Caps (Strips of 8 Cap Mats)
Caps recommended for use with the 600 µL tubes for direct injection right after labeling and SPE.
15 cap strips of 8
1 mL Round Collection Tray (96-well)
Fitted for the vacuum manifold and 600 µL tubes. Place 600 µL tubes directly in for collection of eluate.
1 tray
Waste Tray For collection of effluent during SPE conditioning, loading, and washing steps. Disposable but can be re-used if desired.
1 tray
Optional PN’s Included in Starter Kits
Ammonium Formate Solution (Mobile Phase A Concentrate) – comes in all Starter Kits
5050 mM Ammonium Formate Concentrate already pH’d for convenience. This can be poured directly into a 1 L LCMS grade bottle of water to get the recommended concentration of 50 mM Ammonium Formate pH 4.4.
1 vial
Table 1. Kit Contents
Kit format: 4 sets of 24 reactions can be completed at a time for a total of 96 samples.
Some products may be classified as hazardous and are intended for use by professional laboratory personnel trained in the competent handling of such materials. Responsibility for the safe use of products rests entirely with the purchaser and user. The Safety Data Sheet (SDS) for this product is available at www.waters.com/sds.
II. STORAGE AND STABILIT YGlycoWorks RapiFluor-MS Kit Components Storage Checklist
Standard/Reagent Initial Storage After Reconstitution Source
Intact mAb Check Standard Freeze on arrival (-20 °C)
10 °C for 1 week or sub-aliquot and freeze. AVOID freeze/thaw cycles.
Intact mAb Mass Check Standard Care and Use Manual (720004420en)
GlycoWorks RapiFluor-MS Performance Test Standard (Optional PN )
Freeze on arrival (-20 °C)
4–10 °C for 1 week or sub-aliquot and freeze (-80) for 3 months.AVOID freeze/thaw cycles.
RapiFluor-MS Performance Test Standard Care and Use Manual (720005349en)
GlycoWorks Rapid PNGase F and GlycoWorks Rapid Buffer
Fridge (2–8 °C )
Rapid PNGase F and GlycoWorks Rapid Buffer should be stored at 2–8 °C for up to 24 months.AVOID freeze/thaw cycles. Bring to room temperature and centrifuge before use.
GlycoWorks RapiFluor-MS Kit Care and Use Manual (715004793en)
RapiGest SF Room temperature
Once reconstituted in high purity water or a buffer (pH 7–10) the solution is stable for one week when stored at 2–8 °C.
Long term storage of frozen aliquots is possible but not recommended due to potential solubilization issues of RapiGest SF or storage buffer.
Note: RapiGest SF hydrolyzes in acidic solutions (half life 8 min. at pH 2 and 60 min. at pH 3).
RapiGest Care and Use Manual (715000122en)
RapiFluor-MS Label Room temperature
It is recommended to store the plastic paraffin film vial in a sealed bag along with a desiccant pack at room temperature.
Once solubilized, the RapiFluor-MS reagent solution should be stored at -80 °C (the freezing point of DMF is -61 °C) under anhydrous conditions. It is suggested to store RapiFluor-MS reagent solution as 12 μL aliquots in 600 μL microcentrifuge tubes and to subject labeling reagent aliquots to only a single freeze-thaw cycle. In order to further minimize contamination of the reagent solution with water condensation, the analyst must allow aliquots to equilibrate to room temperature before the aliquot tube is opened.
GlycoWorks RapiFluor-MS Kit Care and Use Manual(715004793en)
GlycoWorks Reagent Solvent Anhydrous DMF Packaged with Label
Room temperature
Once the ampoule is opened, it needs to be used right away to solubilize the label.
GlycoWorks RapiFluor-MS Kit Care and Use Manual(715004793en)
GlycoWorks SPE Reagents (SPE Elution Buffer and Sample Diluent)
Room temperature
Stable until expiration date. If opened many times, it is good to check pH of elution buffer. Once SPE Diluent is opened use it right away to avoid ACN evaporation to minimize chromatography solvent effects.
GlycoWorks RapiFluor-MS Kit Care and Use Manual(715004793en)
III. USING THE GLYCOWORKS RAPIFLUOR-MS N -GLYCAN KIT The GlycoWorks RapiFluor-MS N-Glycan Kit - 96 sample is designed to provide scientists with all materials needed to successfully perform
fast N-glycan deglycosylation, rapid fluorescent labeling, and glycan extraction with minimal preparation time.
Table 2. Checklist of Additional Materials for Sample Preparation Needed Before You Begin
Material Description* Recommended Suppliers
Formic Acid LC-MS-grade formic acid is recommended, Fisher p/n A117 or equivalent.
18.2 MΩ Water For glycan sample prep. LC-MS-grade water is critical for glycan analysis, Fisher p/n W6 or equivalent.
Acetonitrile LC-MS-grade acetonitrile is highly recommended for mass spectrometry analysis of glycans, Fisher p/n A955 or equivalent.
Ammonium Formate Solution Recommended use is Waters (p/n 186007081), or use Fisher p/n A115.
96-well Plate Extraction Vacuum Manifold
SPE Vacuum Pump
Vacuum Manifold Shims (set of 3)
To process the sample via µElution SPE, use either a vacuum manifold or a positive pressure manifold.
The SPE steps of this protocol will require the use of shims for optimal positioning of components in a vacuum manifold.
Positive Pressure Processor
(or) Positive Pressure Processor Spacer
To process sample via µElution SPE, use either a vacuum manifold or a positive pressure processor.
When processing the SPE plate using a positive pressure processor, a specially designed spacer is required.
Heat Block/Thermocycler This protocol requires the user to have either two separate calibrated dry heating baths capable of heating <100 µL volumes at 50 and 90 °C, respectively, or a thermocycler with rapid, programmable temperature changes. A custom heat block (p/n 186007985) was designed to hold the included 1 mL tubes that are in strips of 8/96 well format. The heat block has been tested to fit into the following dry bath heaters. Comparable Dry Block Heaters include: Boekel: Model 112002 for 115 V AC (American power plug) and Model 112002-2 for 230 V AC (Continental European power plug), VWR 2 Block Heater – VWR p/n 12621-058, Fisher Scientific Dry Block Heater – Fisher p/n 88860022, Thermo Scientific Dry Block Heater Fisher PN 11-720-10BQ or the Boekel 2 Block heater – VWR p/n 63999-202.
Centrifugal Vacuum Evaporator May be needed to concentrate sample prior to LC or LC-MS analysis (optional).
Pipettes 1–10 µL, 10–100 µL, 20–200 µL, and 100–1,000 µL capacity. For higher throughput SPE, 20–200 µL and 100–1,000 µL capacity 8-channel pipettes can be used.
*Part numbers of available accessories can be found at the end of this document.
Glycoworks HILIC µElution Plate
Room temperature-dry
After partial use store in the open pouch, squeeze out any air, fold over the open end of the pouch and seal with tape. Store in a desiccator.
GlycoWorks RapiFluor-MS Kit Care and Use Manual(715004793en)
Ammonium Formate Solution (Mobile Phase A Concentrate) – (Starter Kits or Optional PN )
Room temperature
Room temperature for no longer than one month once solubilized. When not in use store it in the fridge – ensuring time to get to room temp upon before re-use.
For smaller volume needs, one could use 1 mL at a time to add to 100 mL volumes of water.
Product COA
ACQUITY UPLC Glycan BEH Amide Column
Room temperature
For column regeneration and storage: Instead of using 100% aqueous mobile phase for regeneration, a 75/25 composition can be used as a means to extend the column lifetime. However, it should be kept in mind that some applications may require a 100% aqueous regeneration step in order to minimize sample derived buildup.
Common chemicals and solvents are not included. Prior to beginning this protocol, the user will need to prepare the following solutions for
the generation of 24 samples for the SPE cleanup module:
1. 5 mL of 18.2 MΩ water
2. 9 mL of acetonitrile
3. 5 mL of 15:85 (v/v) 18.2 MΩ water/acetonitrile
4. 50 mL of 1:9:90 (v/v/v) formic acid/18.2 MΩ water/acetonitrile
Note: Volumes should be measured out individually and accurately (± 3%)
and then combined to make solutions 3 and 4 for the cleanup step.
1. The recommended glycoprotein concentration is 2 mg/mL.
A volume of 7.5 μL of glycoprotein solution is to be used in this
procedure. Glycoprotein samples of different concentration can
be accommodated by adjusting the water volume in Step 6.
Please note that this protocol is designed for a glycoprotein
quantity of 15 µg. Changes to the glycoprotein quantity will
affect the PNGase F to substrate ratio as well as the molar
excess of labeling reagent, which will potentially result in low
yield or so-called over labeling artifacts (see the tips and tricks
of Step 2 for more information).
2. Use of a control standard is highly recommended. Intact mAb
Mass Check Standard is provided. Reconstitute 1 vial
(1 mg/vial) of Intact mAb Mass Check Standard in 500 μL
of 18.2 MΩ water to create a 2 mg/mL IgG solution.
3. Prepare a buffered solution of 5% (w/v) RapiGest by dissolving
the contents of 1 vial (10 mg) of RapiGest in 200 μL of
5x GlycoWorks Rapid Buffer (found in the box with the
GlycoWorks Rapid PNGase F enzyme). Vortex to mix.
4. Add 15.3 μL of 18.2 MΩ water into a 1 mL tube.
5. Dispense 7.5 μL of the 2 mg/mL glycoprotein solution into
the provided Sample Collection Module box. Deglycosylation
Reaction: 1 ml Tubes or equivalent. Deglycosylation Reaction
Caps are included but optional.
6. Add 6 μL of buffered solution containing 5% (w/v) RapiGest SF.
Aspirate and dispense to mix.
nn Each reaction in the GlycoWorks RapiFluor-MS N-Glycan Kit is optimized for the release, labeling and extraction of N-glycans from 15 µg of glycoprotein. PNGase F and RapiFluor-MS reagent concentrations are designed for this exact quantity of sample. The GlycoWorks RapiFluor-MS N-Glycan Kit is designed for nucleophile free samples.
nn RapiGest SF is an anionic surfactant used in this enzymatic protocol to ensure that N-glycans are accessible to Rapid PNGase F, and that upon heat denaturation, glycoproteins do not precipitate out of solution. RapiGest is an enzyme-friendly reagent and can therefore be used at high concentrations alongside Rapid PNGase F.
nn Once RapiGest SF is reconstituted in the Rapid PNGase F Buffer as a 5% (w/v) solution, it should be used within a day. The 5% (w/v) solution of RapiGest SF can be stored at -80˚C for up to one month if longer term storage is needed between sample preparations.
nn This kit was designed to work in conjunction with HEPES buffer. The Rapid PNGase F Buffer is a 5x stock solution comprised of 250 mM HEPES and has been titrated with NaOH such that upon dilution to a 1x solution a pH of 7.9 is obtained.
beginning so that solutions will be heated to 50 °C and at least 90 °C.
It may take 10 to 30 minutes for the heat blocks to equilibrate.
Buffer/Formulation Considerations: Avoid nucleophiles and SDS. The rapid deglycosylation procedure is facilitated by the RapiGest SF Surfactant and will be comprised by the presence of SDS. Likewise, rapid labeling will be compromised by the presence of high concentrations of nucleophiles. It is advised to dilute amine and/or thiol concentrations to <0.1 mM. Some samples may therefore require a buffer exchange step before enzymatic deglycosylation. Finally, other protein content in a sample (such as an albumin excipient) must be considered as contributing to the 15 µg quantity of protein that is prepared by each reaction in this kit.
To perform a buffer exchange or to prepare samples out of complex matrices (lysates/biofluids), consider the use of molecular weight cut off (MWCO) filtration, dialysis, or protein precipitation (i.e. ethanol precipitation). With these techniques, it is recommended to exchange a protein sample into a neutral sodium phosphate, citrate, or HEPES buffer. A matching formulation to the GlycoWorks Rapid Buffer would be 50 mM HEPES free acid titrated to pH 7.9 with sodium hydroxide.
Avoid SDS and Nucleophiles
Tris DTT Glycine HistidineTrTT is ycine tidine
Ammonium Mercaptoethanol
nn Bring PNGase F up to room temperature and centrifuge before use.
Rapid deglycosylation method with Rapid PNGase F, Rapid Buffer, 1% RapiGest SF plus 4 mM TCEP.
It is suggested that TCEP be used rather than a thiol-based, nucleophilic reducing agent, such as DTT. TCEP can be tolerated in the final deglycosylation mixture at low concentrations without causing diminished Rapid PNGase F activity or negative effects on subsequent rapid labeling. TCEP HCl is an acidic salt, so care must be taken to neutralize a TCEP HCl solution with NaOH prior to use in this application. In addition to these conditions, extended incubation at this step, for up to one hour, can help.
Specialized, in-house adaptation
Extraordinarily challenging samples to completely deglycosylate
Rapid labeling with GlycoWorks RapiFluor-MS and subsequent GlycoWorks HILIC SPE can be matched to specialized deglycosylation techniques. It is advised to not deplete protein from samples prior to labeling. RapiFluor-MS labeling reactions should be performed at reagent concentrations of 36 mM and glycoprotein concentrations of 0.36 mg/mL. It is important to maintain the molar excess of the above conditions if glycoprotein concentration is adjusted.
7. Heat denature this mixture for 3 minutes using a heat block
such that the solution temperature reaches at least 90 °C.
nn These conditions can be achieved by heating the 1 mL tube with the customized heat block (p/n 186007985) for 3 minutes with the heat setting turned to ~8.5 to 9 or to a measured heat block surface temperature of 105–110 °C. The maximum temperature achieved during this heat denaturation can be a critical factor in this sample preparation. It is good practice to calibrate the settings of your instrument beforehand.
nn It is not necessary to cap the tube during this and subsequent incubation steps nor is it necessary to centrifuge any condensate that forms at the top of the tube prior to proceeding to the next steps.
nn Incubation can optionally be performed with a thermocycler. In which case, it is likely that 200 µL PCR tubes will be used. This protocol is adaptable to this vessel, though it will be necessary to transfer the glycan mixture to a ≥1 mL tube for dilution with ACN immediately before HILIC SPE (Step 2, 5). Alternatively, deglycosylation and labeling can be carried out at 2x concentrations such that sample transfer is not required upon ACN dilution (See Figure 6 for guidelines on using the GlycoWorks RapiFluor-MS N-Glycan Kit in this manner).
nn This can be accomplished by heating the 1 mL tube in an analog Boekel dry block heater with the heat setting turned to ~5.5 or to a measured heat block surface temperature of 57 °C.
For customers using a thermocycler
Component/stepStandard protocol (1 mL tube)
Protocol adapted for use of 200 μL PCR tubes
Step 1 2 mg/mL antibody sample 7.5 μL 7.5 μL5% (w/v) RapiGest SF in GlycoWorks Rapid Buffer
6 μL 3 μL
Water 15.3 μL 3.3 μLHeat denaturation (at least 90 °C) and cool downGlycoWorks Rapid PNGase F 1.2 μL 1.2 μLDeglycosylation total volume 30 μL 15 μL
Labeling at room temperatureACN dilution 358 μL 179 μLTotal volume of the ACN-diluted, labeled sample
400 μL 200 μL
8. Remove the 1 mL tube from the heat block, allowing it to cool for 3 minutes.
9. Add 1.2 μL of GlycoWorks Rapid PNGase F, enzyme bringing the IgG concentration to 0.5 mg/mL. Aspirate and dispense to mix.
10. Incubate this mixture such that the solution temperature is maintained at 50 °C for 5 minutes. During this incubation, prepare the labeling reagent solution (Step 2, page 8).
11. Remove the 1 mL tube from the heat block, allowing the deglycosylation mixture to cool at room temperature for 3 minutes.
It is imperative that the glycoprotein be subjected to heat denaturation prior to the addition of PNGase F. In the heat denaturation step, ensure that the glycoprotein is subjected to a temperature of at least 90 °C. Some challenging samples may require such a high temperature and possibly even near-boiling conditions (100 °C) in order for complete deglycosylation to be achieved.
Quality Control and Automation Friendly GlycoWorks RapiFluor-MS
and dispense the solution 5–10 times to ensure the reagent is
dissolved. Alternatively, cap and briefly vortex the contents of
the reagent vial.
2. Add 12 μL of the RapiFluor-MS Reagent Solution to the
deglycosylation mixture contained in the 1 mL tube.
3. Aspirate and dispense the reagent solution 5 times to
ensure mixing.
4. Allow the labeling reaction to proceed at room temperature.
5. After 5 minutes, dilute the reaction with 358 μL of
ACN in preparation for HILIC SPE.
nn RapiFluor-MS is purified as a 1:1 complex with NHS. The formula weight for the reagent as provided is 542.41 g/mol.
nn RapiFluor-MS is a highly reactive, primary/secondary amine labeling reagent. It hydrolyzes in water with a half life on the order of 10–100 seconds (see Figure 4). It is, therefore, important that the reagent be dissolved in the provided anhydrous DMF, a non-nucleophilic, polar aprotic solvent. Reagent solution can be used across the span of a day if care has been taken to limit exposing the solution to atmospheric moisture. The GlycoWorks RapiFluor-MS N-Glycan Kit is segmented into 24 reaction sets so that fresh reagent can be used whenever possible.
nn Glycans are released from glycoproteins as glycosylamines, an important fact for an analyst to consider when using this labeling chemistry.
nn The concentrations of reagent and DMF solvent specified in this protocol have been optimized to ensure high yield of glycosylamine labeling. It is not recommended to make adjustments to these values without considering the yield and/or selectivity of the labeling. It is suggested to maintain a ratio of the RapiFluor-MS reagent weight concentration to protein weight concentration of ~50, so long as labeling reactions are performed on deglycosylation mixtures that have not been depleted of protein matter. DMF is present as a co-solvent in this reaction at a concentration of ~30% to ensure that RapiFluor-MS remains soluble throughout the labeling step.
nn RapiFluor-MS is an NHS-carbamate reagent. Unlike NHS-ester reagents, it hydrolyzes over time to generate carbon dioxide and a corresponding amine. This makes RapiFluor-MS labeling a self-quenching reaction. If the reaction is allowed to proceed at room temperature for a minimum of 5 minutes, a quenching step need not be performed.
RapiFluor-MS Reagent Powder
O NH
ON
O
NHN
N
O
OOH
N
O
O
+
Rapid Fluorescence
MS
+
+
H2O
+ + CO2
Figure 4. Reaction schematic for RapiFluor-MS derivatization of an N-glycosylamine. The pathway on the left shows the derivatization of a glycosylamine, which produces an N-glycan with a urea (NH-CO-NH) linked RapiFluor-MS label. Hydrolysis of RapiFluor-MS is shown in the pathway on the right.
Step 3. HILIC SPE Clean-Up of Labeled Glycosylamines
1. Set up a GlycoWorks HILIC µElution Plate on a vacuum manifold
outfitted with a set of 3 shims (See Table 2) and the waste tray
included in the Sample Collection Module box.
Waters has developed short, 2-minute videos for the proper use of Vacuum Manifold and Positive Pressure Processor. To watch the videos visit www.waters.com/manifolds.
2. Condition wells to be used on the μElution plate with 200 μL
of 18.2 MΩ water.
3. Equilibrate wells with 200 μL of 15:85 water/acetonitrile.
4. Load the acetonitrile diluted samples in their entirety ~400 µL.
5. Wash the well with two (2) 600 μL volumes of 1:9:90 (v/v/v)
formic acid/water/acetonitrile.
6. Replace the waste tray with a 96-well collection plate fitted
with the Labeling Reaction: 600 µL Tubes included in the
GlycoWorks Sample Collection Module box.
7. Elute glycans with three (3) 30 μL volumes of GlycoWorks SPE
Elution Buffer (200 mM ammonium acetate in 5% acetonitrile).
nn The tapered bottom inserts are provided in an 8-strip format. Separate tubes as needed for your workflow and throughput.
nn To improve uniformity of flow across different wells- one can condition 2x with water.
nn This elution buffer has been developed to deliver optimal, unbiased recovery of both small neutral glycans as well as high molecular weight, heavily sialylated glycans.
Step 4. Preparing Labeled Glycans for HILIC-FLR with an ACQUITY UPLC BEH Glycan Column
1. Dilute the 90 μL eluate with 310 µL of the GlycoWorks Sample
Diluent-DMF/ACN included in the kit prior to HILIC chromatography.
Aspirate and dispense 5x to ensure complete mixing.
nn Sample is stable at 4 °C for at least 3 days. If sitting for more than a day, before injection vortex to ensure proper mixing to minimize solvent effects if any condensation occurred. For long term storage, freeze at -80 °C.
2. Cap the tapered bottom insert using the provided pre-slit,
strip-format Labeling Reaction Caps included in the
GlycoWorks Sample Collection Module box.
nn Sample can be prepared for analysis from the SPE eluate in multiple ways. It is suggested to perform direct analysis of DMF/ACN diluted eluate. DMF is used as a co-solvent in the sample diluent to ensure miscibility and to minimize loss of sample due to precipitation. DMF/ACN dilution as described in this step allows injection volumes to be as large as 30 µL when a 2.1 mm I.D. column is used. Alternatively, the SPE eluate can be dried by centrifugal vacuum evaporation, reconstituted in water, and injected at a volume of ≤1 μL when a 2.1 mm I.D. column is used. If the above recommendations are followed, the analyst will not encounter solvent effects, such as peak broadening and peak splitting.
nn The cap mats are provided in an 8-strip format to match to the 8-strip format of the 600 µL inserts. Cut smaller lengths of the 8-strip mat as needed for your experiments.
nn GlycoWorks SPE can also be accomplished using a positive pressure manifold (PPM) outfitted with a PPM Spacer (See Table 2).
nn General Guidelines for Vacuum Manifold: A vacuum setting of 2.5–4 in Hg is optimal for all steps in the SPE process. To pull higher vacuum on isolated wells, one can cover un-used wells.
nn General Guidelines for Positive Pressure Manifold: For loading, washing, and elution, approximately 3 psi is appropriate. For conditioning, 10–20 psi can be used.
nn For low sensitivity mass spectrometers: The GlycoWorks RapiFluor-MS N-Glycan Kit was designed to provide a sample for direct LC-MS analysis using a mass spectrometer built with StepWave™ Technology. These instruments, such as an ACQUITY® QDa®, Xevo® G2-XS QTof, and SYNAPT® G2-Si HDMS, exhibit the levels of sensitivity needed to facilitate glycan LC-MS. For lower sensitivity mass spectrometers, it is suggested to dry the obtained 90 µL SPE eluate and to reconstitute the sample sequentially with 4.5 µL of water, 5 µL of DMF, and finally 10.5 µL of ACN. This highly concentrated sample can then be injected in its entirety to increase MS signal as needed.
Care & Use ManualsRapiFluor-MS Dextran Ladder Calibration Care and Use Manual 720005348EN
RapiFluor-MS Glycan Performance Test Standard Care and Use Manual 720005349EN
96-well Extraction Plate Map 720004758EN
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm, Columns and Glycoprotein Performance Test Standard 720005408EN
XBridge Glycan BEH Amide XP, 130Å, 2.5 μm and 3.5 μm Columns and Standards 720004882EN
ACQUITY UPLC BEH Glycan, 1.7 µm Columns and Glycan Performance Test Standard 720003042EN
RapiFluor-MS High Mannose Test Standard 720005531EN
RapiFluor-MS Sialylated Glycan Performance Test Standard 720005778EN
RapiFluor-MS Quantitative Test Standard 720005917EN
Application Notes and PapersLauber MA, Koza S, Fountain KJ. Optimization of HILIC SPE for the Quantitative and Robust Recovery of N-Linked Glycans 720004717EN
Lauber MA, Brousmiche DW, Hua Z, Koza S, Guthrie E, Magnelli P, Taron CH, Fountain KJ. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Novel Fluorescence and MS-Active Labeling Reagent
Transferring RapiFluor-MS Labeled N-Glycan HILIC Separations Between UPLC and HPLC 720005344EN
New Capabilities for Monitoring Released N-Glycans through the Combined Use of RapiFluor-MS Labeling, ACQUITY UPLC H-Class Bio System, and Serial Fluorescence/ACQUITY QDa Mass Detection
720005352EN
Exploiting RapiFluor-MS Labeling to Monitor Diverse N-Glycan Structures via Fluorescence and Mass Detection 720005353EN
Robustness of RapiFluor-MS N-Glycan Sample Preparations and Glycan BEH Amide HILIC Chromatographic Separations 720005370EN
Profiling Released High Mannose and Complex N-Glycan Structures from Monoclonal Antibodies Using RapiFluor-MS Labeling and Optimized Hydrophilic Interaction Chromatography
720005516EN
Quality Control and Automation Friendly GlycoWorks RapiFluor-MS N-Glycan Sample Preparation 720005506EN
Applying a Novel Glycan Tagging Reagent, RapiFluor-MS, and an Integrated UPLC-FLR/QTof MS System for Low Abundant N-Glycan Analysis
720005383EN
Characterization of EPO N-Glycans using RapiFluor-MS and HILIC Profiling 720005444EN
Rapidly Monitoring Released N-Glycan Profiles during Process Development Using RapiFluor-MS and the ACQUITY QDa Detector 720005438EN
Transferring RapiFluor-MS Labeled N-Glycan HILIC Separations Between UPLC and HPLC 720005344EN
Comprehensive Characterization of the N and O-Linked Glycosylation of a Recombinant Human EPO 720005462EN
Enhancing the Peak Capacity of High Molecular Weight N-Glycan HILIC Separations with a Wide-Pore Amide Bonded Stationary Phase
720005381EN
RapiFluor-MS Facilitates Versatile Detection of Released N-Glycans 720005646EN
Absolute Quantitation of RapiFluor-MS Labeled N-Glycans by Calibration of Fluorescence Response 720005981EN
Optimizing HILIC-based Analyses of RapiFluor-MS Labeled Sialylated N-Glycans 720005850EN
Combining RapiFluor-MS and UNIFI Software for a Total N-linked Glycan Solution for Comparing Innovator vs. Biosimilar Infliximab 720005753EN
* Search for documents by literature code on waters.com
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
Waters, The Science of What’s Possible, ACQUITY, UPLC, QDa, Xevo, UNIFI, MassLynx, XBridge, Empower, GlycoWorks, RapiFluor-MS, and SYNAPT are registered trademarks of Waters Corporation. StepWave, μElution, and RapiGest are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
