[ CARE AND USE MANUAL ]
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
CONT ENT S
I.INT RODUCTION
II. STORAGE AND STABILIT Y
III. USING THE GLYCOWORKS
RAPI FLUOR-MS N-GLYCAN KIT
IV. ORDERING INFORMATION
However, the user is advised to confirm enzymatic release for their
particular sample. This innovative sample preparation uses optimized
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.
V.G LYCOWO RK S RAPI F LUO R-MS
QUIC K STA RT 8 SAM P L E P ROTOCOL
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.
VI. REFERENCES AND
ADDITIONAL RESOURCES
RapiFluor-MS Reagent
AccQ·Fluor
VII. COMMON GLYCANS NAMES, MASSES,
AND CHARGE STAT ES
Rapid
Fluorescence
RapiFluor-MS
O
O
N
I.INT RODUCTION
This document provides information regarding the general care and use
of the Waters GlycoWorks™ RapiFluor-MS™ N-Glycan Kit – 24 sample
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.
Figure 1. GlycoWorks RapiFluor-MS Start Up Kit – 24 sample.
N
O
O
Rapid
N
MS
NH
Tertiary Amine
Charge Tag
O
NHS Carbamate Rapid
Tagging Group
NH
Fluorescence
Quinolinyl
Fluorophore
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.
[ CARE AND USE MANUAL ]
Streamlined Protocol for a Simple and Fast Sample Preparation that Minimizes Sample Handling and Loss
Glycoprotein
STEP 1: De
Deglycosylation
egl cos
os lation
osylation
< 15 minutes
Released N-Glycans
ycans
+ Protein
O
O
O
STEP 2: Labeling
N
O
N
O
NH
N
+
N
O
NH
O
5 minutes
Labeled
Reaction
on
N-Glycans
Byproducts
ucts
+ Reagents
STEP 3: Clean-up
10 minutes
in as little as
30 minutes
Labeled Glycans
Analysis
LC-FLR
LC-FLR-MS
Figure 3. Workflow for the rapid preparation of N-glycans using the RapiFluor-MS N-Glycan Kit.
2
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
OH
Table 1. Kit Contents
Kit format: 3 sets of 8 reactions can be completed at a time for a total of 24 samples.
Kit Component
Purpose
Quantity in Kit
GlycoWorks Deglycosylation Module
Intact mAb Mass Check Standard
1 mg of an intact mouse IgG1 monoclonal antibody for use as a control for
process validation and troubleshooting.
1 vial
GlycoWorks Rapid PNGase F and
Buffer
GlycoWorks Rapid PNGase F (Kit Component) = 0.03 mL of PNGase F enzyme
for use with rapid deglycosylation procedures. Recombinant source.
1 vial
GlycoWorks Rapid Buffer
GlycoWorks Rapid Buffer (0.25 mL of 250 mM HEPES pH 7.9)
For use with deglycosylation/labeling.
1 vial
RapiGest™ SF
An enzyme-friendly surfactant used to denature the glycoproteins and to facilitate
the deglycosylation reaction. Each set of 24 reactions requires one 3 mg vial.
3 vials
GlycoWorks RapiFluor-MS Labeling Module
GlycoWorks Rapi Fluor-MS
Reagent Powder
9 mg/vial of Rapi Fluor-MS reagent is included for labeling 8 samples.
3 vials
GlycoWorks Reagent Solvent
Anhydrous DMF
1 mL ampoule of anhydrous dimethylformamide (DMF) solvent is used
for solubilization of the RapiFluor-MS reagent.
3 ampoules
GlycoWorks Clean-up Module (Includes SPE plate and reagents in 2 separate boxes)
GlycoWorks HILIC μElution Plate
For removing potential interferences like labeling reaction byproducts
from the RapiFluor-MS labeled glycans and excess label.
1 plate
The 5 mg sorbent was tested and found to have maximum binding capacity
for 200 μg glycans.
GlycoWorks SPE Reagents
GlycoWorks SPE Elution Buffer
0.5 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
3 mL/vial DMF solvent 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
50 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
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.
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
3
[ CARE AND USE MANUAL ]
II. STORAGE AND STABILIT Y
GlycoWorks 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.
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.
RapiGest Care and Use Manual
(715000122en)
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).
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.
GlycoWorks RapiFluor-MS Kit
Care and Use Manual
(715004793en)
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 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.
GlycoWorks RapiFluor-MS Kit
Care and Use Manual
(715004793en)
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.
Product COA
For smaller volume needs, one could use 1 mL at a
time to add to 100 mL volumes of water.
4
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
III. USING THE GLYCOWORKS RAPI FLUOR-MS N-GLYCAN KIT
The GlycoWorks RapiFluor-MS N-Glycan Kit – 24 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
To process the sample via µElution SPE, use either a vacuum manifold or a positive pressure manifold.
SPE Vacuum Pump
The SPE steps of this protocol will require the use of shims for optimal positioning of components in a
vacuum manifold.
Vacuum Manifold Shims (set of 3)
Positive Pressure Processor
To process sample via µElution SPE, use either a vacuum manifold or a positive pressure processor.
(or) Positive Pressure Processor Spacer
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 RapiFluor-MS N-Glycan Kit – 24 Sample
5
[ CARE AND USE MANUAL ]
Getting Started:
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 8 samples for the SPE cleanup module:
1. 2 mL of 18.2 MΩ water
2. 3 mL of acetonitrile
3. 2 mL of 15:85 (v/v) 18.2 MΩ water/acetonitrile
4. 20 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.
Step 1: Rapid Deglycosylation:
GlycoWorks Deglycosylation Module
Before you start: It is recommended if using
heat blocks to set the temperatures prior to
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.
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 (3 mg) of RapiGest in 60 μL of
5x GlycoWorks Rapid Buffer. 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 1 mL tube.
6. Add 6 μL of buffered solution containing 5% (w/v) RapiGest SF.
Aspirate and dispense to mix.
6
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
Avoid SDS and Nucleophiles
Buffer/Formulation Considerations:
Avoid nucleophiles and SDS. The
TTris
Tr
is
DTT
Glycine
ycine
Histidine
tidine
rapid deglycosylation procedure
is facilitated by the RapiGest SF
Ammonium
Mercaptoethanol
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.
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.
7. Heat denature this mixture for 3 minutes using a heat block
such that the solution temperature reaches at least 90 °C.
For customers using a thermocycler:
Standard
protocol
(1 mL tube)
Component/step
Step 1
Step 2
2 mg/mL antibody sample
5% (w/v) RapiGest SF in
GlycoWorks Rapid Buffer
Water
Heat denaturation
GlycoWorks Rapid PNGase F
Deglycosylation
total volume
Incubate for deglycosylation
Protocol adapted
for use of 200 μL
PCR tubes
7.5 μL
7.5 μL
6 μL
3 μL
15.3 μL
3.3 μL
1.2 μL
1.2 μL
30 μL
15 μL
nn These conditions can be achieved by heating the 1 mL tube in an
analog Boekel dry bath heater with a matching heat block 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
GlycoWorks RapiFluor-MS
9 mg in
Reagent Reconstitution
131 μL DMF
GlycoWorks RapiFluor-MS
12 μL
Reagent Solution
Labeling at room temperature
ACN dilution
358 μL
Total volume of the ACN400 μL
diluted, labeled sample
9 mg in
66 μL DMF
6 μL
179 μL
200 μL
Quality Control and Automation Friendly GlycoWorks RapiFluor-MS N-Glycan
Sample Preparation (720005506EN).
8. Remove the 1 mL tube from the heat block, allowing it
to cool for 3 minutes.
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).
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.
9. Add 1.2 μL of Rapid PNGase F, 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).
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.
11. Remove the 1 mL tube from the heat block, allowing the
deglycosylation mixture to cool at room temperature for 3 minutes.
Deglycosylation method development/troubleshooting
Workflow
Standard rapid
deglycosylation
workflow
Sample
Complete
deglycosylation of
most glycoproteins
Recommended method
Rapid deglycosylation method with Rapid PNGase F, Rapid Buffer, and 1% RapiGest SF.
Adapted rapid
deglycosylation
workflow
Challenging
glycoprotein
possessing
disulfide bonds
Rapid deglycosylation method with Rapid PNGase F, Rapid Buffer, 1% RapiGest SF plus 4 mM TCEP.
Specialized,
in-house
adaptation
Extraordinarily
challenging samples
to completely
deglycosylate
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.
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.
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
7
[ CARE AND USE MANUAL ]
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.
O
O
O
N
O
N
O
+
N
MS
NH
O
Rapid
NH
Fluorescence
N
OH
O
RapiFluor-MS Reagent Powder
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.
Step 2: Rapid Labeling of Glycosylamines
1. Prepare reagent solution by dissolving one (1) vial of 9 mg
of RapiFluor-MS in 131 μL of anhydrous DMF. Aspirate and
nn Glycans are released from glycoproteins as glycosylamines, an important
dispense the solution 5–10 times to ensure the reagent is
fact for an analyst to consider when using this labeling chemistry.
dissolved. Alternatively, cap and briefly vortex the contents
of the reagent vial.
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.
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.
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 selfquenching reaction. If the reaction is allowed to proceed at room
temperature for a minimum of 5 minutes, a quenching step need not
be performed.
5. After 5 minutes, dilute the reaction with 358 μL of
ACN in preparation for HILIC SPE.
+
Mass shift from glycosylamine
+C17H2ON4O2 (ΔMi = 312.1586 Da/ΔMAvg = 312.3663 Da)
H2O
Mass shift from glycans with free reducing end
+C17H21N5O1 (ΔMi = 311.1746 Da/ΔMAvg = 311.3815 Da)
+
+
+
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.
8
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
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 a waste tray.
nn GlycoWorks SPE can also be accomplished using a positive pressure
manifold (PPM) outfitted with a PPM Spacer (See Table 2).
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.
nn General Guidelines for Vacuum Manifold: A vacuum setting
2. Condition wells to be used on the μElution plate with 200 μL
of 18.2 MΩ water.
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,
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 600 µL tapered bottom inserts.
7. Elute glycans with three (3) 30 μL volumes of GlycoWorks
SPE Elution Buffer (200 mM ammonium acetate in
5% acetonitrile).
washing, and elution, approximately 3 psi is appropriate.
For conditioning, 10–20 psi can be used.
nn To improve uniformity of flow across different wells- one
can condition 2x with water.
nn The tapered bottom inserts are provided in an 8-strip format.
Separate tubes as needed for your workflow and throughput.
nn T his elution buffer has been developed to deliver optimal, unbiased
recovery of both small neutral glycans as well as high molecular
weight, heavily sialylated glycans.
nn Sample can be prepared for analysis from the SPE eluate in multiple
Step 4: Preparing Labeled Glycans for HILIC-FLR
with an ACQUITY UPLC BEH Glycan Column
1. Dilute the 90 μL eluate with 100 μL of DMF followed by 210 μL
of ACN prior to HILIC chromatography. Aspirate and dispense
5x to ensure complete mixing.
2. Cap the tapered bottom insert using the provided pre-slit,
strip-format cap mats.
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 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.
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
9
[ CARE AND USE MANUAL ]
Step 5. Recommended Analytical Approach for GlycoWorks RapiFluor-MS-labeled Glycan Analysis
Before You Get Started: Glycan BEH Amide Column Equilibration
When the column is first received, flush in 60% acetonitrile: 40% aqueous (or initial starting conditions) for 50 column volumes.
Equilibrate with 20 column volumes of initial mobile phase conditions before making first injection. Equilibrate with 8–10 column volumes
between injections. Failure to appropriately equilibrate the column could result in drifting retention times. Please refer to the ACQUITY
UPLC® Glycan BEH Amide Column Care and Use (p/n 720003042EN) on www.waters.com for proper use and storage of column.
Universal N-Glycan Profiling Method
Column:
ACQUITY UPLC Glycan BEH Amide, 130 Å, 1.7 µm, 2.1 x 150 mm (p/n 186004742)
Temp.:
60 °C
Mobile phase A:
50 mM ammonium formate solution, pH 4.4 (Use of
the Waters mobile phase concentrate (p/n 186007081)
along with LC-MS-grade water [Fisher p/n W6 or
equivalent] is recommended.)
Mobile phase B:
100% acetonitrile (LC-MS-grade ACN is recommended,
Fisher p/n A955 or equivalent)
Flow rate:
0.4 mL/min
Gradient: Time
(min)
0.0
35.0
36.5
39.5
43.1
47.6
55.0
Flow rate
(mL/min)
0.4
0.4
0.2
0.2
0.2
0.4
0.4
FLR wavelengths:
EX 265/EM 425 nm
FLR sampling rate:
2 Hz
10
%A
25
46
100
100
25
25
25
%B
75
54
0
0
75
75
75
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
Curve
6
6
6
6
6
6
6
Mobile Phase Recommendations
It is critical to use LC-MS-grade solvents and
buffers when performing LC-MS glycan analysis.
Seal wash and LC autosampler wash:
70% LC-MS-grade ACN
30% LC-MS-grade water (v/v)
Injection vol.:
10 µL suggested (≤30 µL, DMF/ACN
diluted SPE eluate, 2.1 mm I.D. columns)
<1 µL (aqueous reconstitution,
2.1 mm I.D. column)
Injector needle:
A stainless steel needle is recommended
when injecting samples containing DMF
as co-solvent.
mAb N-Glycan Profiling Method
LC system:
ACQUITY UPLC H-Class Bio System
Mobile phase B: ACN (LC-MS grade)
Sample temp.: 10 °C
Gradient:
Time
(min)
0.00
3.00
35.0
36.5
39.5
43.1
47.6
Flow rate
(mL/min)
0.5
0.5
0.5
0.2
0.2
0.2
0.5
%A
20
27
37
100
100
20
20
%B
80
73
63
0
0
80
80
Curve
55.0
0.5
20
80
6
Analytical col. temp.: 45 °C
Flow rate:
0.5 mL/min
Injection volume: 10 μL (DMF/ACN-diluted samples),
1 μL (aqueous samples)
Column: ACQUITY UPLC Glycan BEH Amide, 1.7 μm,
2.1 x 150 mm (p/n 186004742)
Fluorescence
detection:
Ex 265 nm/Em 425 nm, 2 Hz
6
6
6
6
6
6
Mobile phase A:
50 mM ammonium formate, pH 4.4
(LC-MS grade; from a 100x concentrate)
(p/n 186007081)
Autosampler Settings for UNIFI,® MassLynx,® and Empower®
UNIFI:
Depending on the version of UNIFI, users
will find the plate is already configured.
Search for “GlycoWorks Plate”, under
Consumables.
Table 3. MassLynx settings for use of the ACQUITY UPLC Autosampler with 600 �L Insert
Vials
Vial spacing (1/10 mm)
Rows
8
90
Columns
12
90
Diameter
Depth
120
370
Vial (1/10 mm)
Plate size (1/10 mm)
X
1280
Y
860
Z
420
Mass Spectrometry Analysis Considerations
Because the RapiFluor-MS label has high proton affinity,
derivatized glycans preferentially adopt high c harge
states during positive ion mode electrospray ionization.
T he predominant charge state for a small neutral glycan
is [M+2H]2+, although it increases to [M+3H]3+ for larger
molecular weight glycans. MS source conditions may require
optimization to ac hieve desired signal, limit in-source
fragmentation, and to minimize adduct formation.
Top left offset (1/10 mm)
X
144
Y
112
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
11
[ CARE AND USE MANUAL ]
Table 4. Empower settings for use of the ACQUITY UPLC
Autosampler with 600 μL Insert Tubes
X
Y
Height
Plate dimensions (mm)
128.00
86.00
42.00
X
Y
Top left well location (mm)
14.40
11.20
Diameter
Depth
7.00
37.00
Well size (mm)
Representative HILIC-FLR-MS data for Intact mAb Mass Check Standard using a Waters SYNAPT G2-S HDMS.
600000.063
FA2
EU x 10e4
EU x 10e4
800000.063
FA2G1
600000.063
400000.031
400000.031
200000.016
A2G1
FA1G1
A2
0.000
9.00
10.00
11.00
12.00
A2G1
13.00
Zoomed View
14.00
15.00
FA2
FA2G2Ga1
16.00
17.00
18.00
19.00
20.00
21.00
22.00
23.00
24.00
FA2G1
800000.063
EU x 10e4
FA2G2Sg1
FA2G2Ga2
FA2G2
FA2G2
600000.063
400000.031
200000.016
A2G1
FA1G1
A2
0.000
9.00
10.00
11.00
12.00
13.00
14.00
FA2G2Sg1
FA2G2Ga2
FA2G2Ga1
A2G1
15.00
16.00
17.00
18.00
19.00
20.00
21.00
22.00
23.00
24.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00
21.00
22.00
23.00
24.00
EU x 10e4
800000.063
m/z
600000.063
RapiFluor-MS
Labeled Glycan
Composition
400000.031
Glycan
200000.016
A2
0.000
9.00
FA2
C67H105O37N9
10.00
11.00
12.00
C73H115O41N9
M i (Da)
1+
1627.6611
1628.6684
13.00
14.00
15.00
1773.7190
1774.7263
m/z
2+
3+
814.8378
16.00
17.00
543.5610
18.00
887.8668
592.2469
M avg (Da)
1628.5887
19.00
20.00
1774.7299
1+
1629.596
21.00
1775.7372
2+
3+
815.30165
22.00
23.00
888.37225
543.8702
Time
24.00
592.58393
FA1G1
C71H112O41N8
1732.6925
1733.6998
867.3535
578.5714
1733.678
1734.6853
867.8463
578.89997
A2G1
C73H115O42N9
1789.7140
1790.7213
895.8643
597.5786
1790.7293
1791.7366
896.37195
597.91707
FA2G1
C79H125O46N9
1935.7719
1936.7792
968.8932
646.2646
1936.8705
1937.8778
969.44255
646.6308
A2G2
C79H125O47N9
1951.7668
1952.7741
976.8907
651.5962
1952.8699
1953.8772
977.44225
651.96393
FA2G2
C85H135O51N9
2097.8247
2098.8320
1049.9196
700.2822
2099.0111
2100.0184
1050.5129
700.67767
FA2G2Ga1
C91H145O56N9
2259.8775
2260.8848
1130.9460
754.2998
2261.1517
2262.159
1131.5832
754.72453
FA2G2Sg1
C96H152O60N10
2404.9150
2405.9223
1203.4648
802.6456
2406.2651
2407.2724
1204.1399
803.09567
FA2G2Ga2
C97H155O61N9
2421.9303
2422.9376
1211.9724
808.3174
2423.2923
2424.2996
1212.6535
808.7714
800000.063
For more detailed characterization of the Intact mAb Mass Check Standard with a RFMS Comparison see “Robustness of RapiFluor-MS
N-Glycan Sample Preparations and Glycan BEH Amide HILIC Chromatographic Separations” (p/n 720005370en).
Mass Spectrometry Analysis Considerations
Because the RapiFluor-MS label has high proton affinity, derivatized glycans preferentially adopt high charge states during positive
ion mode electrospray ionization. T he predominant charge state for a small neutral glycan is [M+2H]2+, although it increases to
[M+3H]3+ for larger molecular weight glycans. MS source conditions may require optimization to achieve desired signal, limit in-source
fragmentation, and to minimize adduct formation.
12
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
IV. O RD E RING INFO RMAT ION
Sample Preparation Complete Kits
Description
Part No.
GlycoWorks RapiFluor-MS N-Glycan Starter Kit – 24 sample
176003712
Kit contains: GlycoWorks Deglycosylation Module (24 sample), GlycoWorks Labeling Module (24 sample), GlycoWorks Clean-up Module, GlycoWorks
Sample Collection Module, ACQUITY UPLC Glycan BEH Amide, 1.7 μm, 2.1 x 150 mm Column, Ammonium Formate Solution – Glycan Analysis
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 sample
Kit contains: GlycoWorks Deglycosylation Module (24 sample), GlycoWorks Labeling Module (24 sample), GlycoWorks Clean-up Module,
GlycoWorks Sample Collection Module
GlycoWorks RapiFluor-MS N-Glycan Basic Kit – 24 sample
176003713
176003911
Kit contains: GlycoWorks Deglycosylation Module (24 sample), GlycoWorks Labeling Module (24 sample), GlycoWorks Clean-up Module.
GlycoWorks RapiFluor-MS N-Glycan Refill Kit – 24 sample
176003714
Refill Kit contains one of each: GlycoWorks Deglycosylation Module (24 sample) and the GlycoWorks Labeling Module (24 sample)
GlycoWorks RapiFluor-MS N-Glycan Starter Kit – 96 sample
Kit contains: GlycoWorks Deglycosylation Module (96 sample), GlycoWorks Labeling Module (96 sample), GlycoWorks Clean-up Module, GlycoWorks
Sample Collection Module, ACQUITY UPLC Glycan BEH Amide, 1.7 μm, 2.1 x 150 mm Column, Ammonium Formate Solution – Glycan Analysis
GlycoWorks RapiFluor-MS N-Glycan Kit – 96 sample
176003635
176003606
Kit contains: GlycoWorks Deglycosylation Module (96 sample), GlycoWorks Labeling Module (96 sample),
GlycoWorks Clean-up Module, GlycoWorks Sample Collection Module
GlycoWorks RapiFluor-MS N-Glycan Basic Kit – 96 sample
176003910
Kit contains: GlycoWorks Deglycosylation Module (96 sample), GlycoWorks Labeling Module (96 sample), GlycoWorks Clean-up Module.
Standards and Accessories
Description
Intact mAb Mass Check Standard**
GlycoWorks RapiFluor-MS Dextran Calibration Ladder
GlycoWorks RapiFluor-MS Glycan Performance Test Standard
GlycoWorks RapiFluor-MS High Mannose Standard
GlycoWorks Rapid Buffer
GlycoWorks Rapid Deglycosylation Kit 1 x 24
GlycoWorks Rapid Deglycosylation Kit 3 x 8
RapiGest SF
RapiGest SF
Ammonium Formate Solution – Glycan Analysis (Mobile Phase Concentrate)
96-Well Plate Extraction Manifold
Vacuum Pump 110/115 V 60H
Vacuum Manifold Shims*
Positive Pressure Manifold
Positive Pressure Manifold Spacer for the GlycoWorks RapiFluor-MS N-Glycan Kit*
Modular Heat Block for 1 mL Tubes/96 Wells
Certified Container Kit
Kit contains: (4) 1 Liter bottles, (3) 500 mL bottles and 1 Cap Kit
*Essential for kit use
Qty.
Part No.
1 mg/vial
50 μg/vial
400 pmol/vial
1,000 pmol/vial
5 mL/vial
1 x 24 reactions
3 x 8 reactions
3 mg/vial
10 mg/vial
50 mM/vial
1
1
3
1
1
1
186006552
186007982
186007983
186008317
186008100
176003867
176003868
186008090
186002123
186007081
186001831
725000417
186007986
186006961
186007987
186007985
1
186007088
**Control Standard included in kit
Columns
Description
ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7 µm, 2.1 x 50 mm Column
ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7 µm, 2.1 x 100 mm Column
ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7 µm, 2.1 x 150 mm Column
XBridge ® Glycan BEH Amide XP, 130Å, 2.5 μm, 3.0 x 150 mm Column
XBridge Glycan BEH Amide XP, 130Å, 2.5 μm, 3.0 x 75 mm Column
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm 2.1 x 50 mm with Std
ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm 2.1 x 100 mm with Std
ACQUITY UPLC Glycoprotein BEH Amide, 300Å , 1.7 μm 2.1 x 150 mm with Std
Glycoprotein Performance Test Standard
Qty.
Part No.
1
1
1
1
1
1
1
1
1
186004740
186004741
186004742
186008040
186008039
176003700
176003701
176003702
186008010
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
13
[ CARE AND USE MANUAL ]
V.G LYCOWO RK S RAPI F LUO R-MS QUIC K STA RT 8 SAM P L E P ROTOCOL
24 Sample Kit: 3 x 8 Format
■■
Set heat blocks so that sample can be heated to 90 °C and 50 °C.
■■
Make solutions for SPE.
■■
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.
STEP 1: Rapid Deglycosylation
1. Prepare 5% (w/v) RapiGest SF by dissolving 3 mg of RapiGest SF Surfactant in
60 μL of Rapid Buffer, vortex.
2. Dispense 15.3 μL of water into a 1 mL tube.
3. Add 7.5 μL of 2 mg/mL glycoprotein into above tube.
4. Add 6 μL of buffered, 5% (w/v) RapiGest SF solution to above tube, aspirate to mix.
5. Heat at least to 90 °C for 3 minutes.
6. Cool at room temperature for 3 minutes.
7. Add 1.2 μL Rapid PNGase F and aspirate to mix.
8. Incubate at 50 °C for 5 minutes.
9. Cool at room temperature for 3 minutes.
STEP 2: Rapid Labeling of Glycosylamines
1. Add 131 μL of anhydrous DMF directly to one vial of 9 mg of RapiFluor-MS reagent.
Mix to solubilize.
2. Add 12 μL of the RapiFluor-MS solution to the deglycosylation mixture and aspirate to mix.
3. Allow the labeling to proceed at room temperature for 5 minutes.
4. Dilute the reaction with 358 μL of acetonitrile (ACN) and aspirate to mix.
STEP 3: HILIC Clean-Up of Labeled Glycosylamines
1. Set up a GlycoWorks HILIC µElution Plate and add in shims or spacer and waste tray.
2. Condition wells by adding 200 μL of water per well.
3. Equilibrate wells by adding 200 μL 85% ACN.
4. Load ACN-diluted samples (~400 μL).
5. Wash wells with two (2) 600 μL volumes of 1% formic acid, 90% ACN.
6. Replace waste tray with sample collection tray loaded with 600 μL tubes.
7. Elute glycans with three (3) 30 µL volumes of SPE Elution Buffer into 600 µL tapered bottom inserts.
8. Dilute SPE eluate with 100 μL of DMF and 210 μL of ACN. Aspirate to mix.
9. Cap the tubes with pre-slit cap mats.
14
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
V I. A DDIT IONA L R E SOU RC E S AND R EF E R ENC E S
Description
Literature Code*
Application Notebooks
Glycan Application Notebook
720005532EN
Videos
Training Video: www.waters.com/RapiFluorMS
Care & Use Manuals
RapiFluor-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
Application Notes and Papers
Lauber 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
720005275EN
Lauber MA, Fournier JL, Koza S, Fountain KJ. GlycoWorks HILIC SPE Robust Glycan Sample Preparation
720005116EN
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
* Search for documents by literature code on waters.com
GlycoWorks RapiFluor-MS N-Glycan Kit – 24 Sample
15
[ CARE AND USE MANUAL ]
V II. COMMON G LYC AN NAM E S, MASS E S, AND C HA RG E STAT E S
Glycan
Oxford notation
Glycan
RapiFluor-MS labeled
glycan composition
Mi (Da)
2+
3+
Mavg (Da)
2+
3+
M5
Man5
C 63 H 99O 37N7
1545.608
773.8113
516.2099
1546.49
774.25
516.5
A2
G0
C 67 H105O 37N 9
1627.6611
814.8378
543.561
1628.59
815.3
543.87
M6
Man6
C 69 H109O 42N7
1707.6609
854.8377
570.2276
1708.63
855.32
570.55
FA1G1
G1F-GN
C 71H112O 41N 8
1732.6925
867.3535
578.5714
1733.68
867.85
578.9
FA2
G0F
C 73 H115O 41N 9
1773.719
887.8668
592.2469
1774.73
888.37
592.58
A2G1
G1
C 73 H115O 42N 9
1789.714
895.8643
597.5786
1790.73
896.37
597.92
FA2G1
G1F
C 79 H125O 46 N 9
1935.7719
968.8932
646.2646
1936.87
969.44
646.63
A2G2
G2
C 79 H125O 47N 9
1951.7668
976.8907
651.5962
1952.87
977.44
651.96
FA2B
G0F+GN
C 81H128 O 46 N10
1976.7984
989.4065
659.9401
1977.92
989.97
660.31
FA2G2
G2F
C 85 H135O 51N 9
2097.8247
1049.9196
700.2822
2099.01
1050.51
700.68
FA2BG1
G1F+GN
C 87 H138 O 51N10
2138.8512
1070.4329
713.9577
2140.06
1071.04
714.36
FA2G1S1
G1F+NANA
C 90 H142O 54 N10
2226.8673
1114.4409
743.2964
2228.13
1115.07
743.72
FA2G2Ga1
G2FGal1
C 91H145O 56 N 9
2259.8775
1130.946
754.2998
2261.15
1131.58
754.72
FA2BG2
G2F+GN
C 93 H148 O 56 N10
2300.9041
1151.4593
767.9753
2302.2
1152.11
768.41
FA2G2S1
G2F+NANA
C 96 H152O 59 N10
2388.9201
1195.4673
797.314
2390.27
1196.14
797.76
FA2G2Sg1
G2F+NGNA
C 96 H152O 60 N10
2404.915
1203.4648
802.6456
2406.27
1204.14
803.1
FA2G2Ga2
G2FGal2
C 97 H155O 61N 9
2421.9303
1211.9724
808.3174
2423.29
1212.65
808.77
FA2BG2S1
G2F+GN+NANA
C104 H165O 64 N11
2591.9995
1297.007
865.0071
2593.46
1297.74
865.49
FA2G2S2
G2F+2NANA
C107 H169O 67N11
2680.015
1341.0148
894.3456
2681.52
1341.77
894.85
FA2BG2S2
G2F+GN+2NANA
C115 H182O 72N12
2883.0949
1442.5547
962.0389
2884.71
1443.36
962.58
Qty: 1
715004903
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Waters, T he Science of W hat’s Possible, ACQUITY UPLC, ACQUITY, QDa, Xevo, Empower, MassLynx, and SYNAPT are registered trademarks
of Waters Corporation. GlycoWorks, RapiFluor, StepWave, and RapiGest are trademarks of Waters Corporation. All other trademarks are the
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©2016 Waters Corporation. Printed in the U.S.A. May 2016 715004903 IH-SIG
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