GlycoWorks RapiFluor-MS N-Glycan Kit – Automation

[ CARE AND USE MANUAL ]

1

CONTENTS

I. INTRODUCTION

II. STORAGE AND STABILITY

III. USING THE GLYCOWORKS RAPIFLUOR-MS N-GLYCAN KIT – AUTOMATION

IV. ORDERING INFORMATION

V. RESOURCES AND REFERENCES

VI. COMMON GLYCAN NAMES, MASSES, AND CHARGE STATES

I. INTRODUCTIONThis document provides information regarding the general care and use of the Waters® GlycoWorks® RapiFluor-MS® N-Glycan Kit - Automation for the fast enzymatic release and rapid labeling of N-glycans, scaled to process 48 or 96 samples at a time. The GlycoWorks RapiFluor-MS N-Glycan Kit – Automation has been designed for use with automated platforms enabling high-throughput glycoprotein sample preparation for analysis of N-glycans with minimal user intervention. This protocol is validated using monoclonal antibodies and has also been tested to perform for a wide range of other N-linked glycoproteins. The user is advised to confirm enzymatic release for their

GlycoWorks RapiFluor-MS N-Glycan Kit – Automation

particular sample especially if any deviation is done relative to workstation requirements and script.

As well as being an automated protocol, this innovative sample preparation uses optimized deglycosylation conditions and reagents for fast release. 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 cleanup prior to LC analysis is accomplished through the use of solid-phase extraction (SPE) with the GlycoWorks HILIC µElution™ Plate.

All reagents in the kit are scaled to residual volumes needed for many automated instrumentation allowing for versatility when choosing your platform. Full automation of 96 samples with script and workstation requirements is typically completed <3 hours when following the provided guidelines and script.

Waters does not support or provide consulting services for automation. If any adaptation is needed based on specific workstation requirements, it is advised to reach out directly to the automation platform vendor for help. If deviations are made to the script or workstation requirements, it is up to the user to ensure they confirm correct results are obtained.

This care and use manual provides highly detailed and informative step-by-step instructions for using this novel protocol. For more information, visit www.waters.com/automate.

Figure 1. GlycoWorks RapiFluor-MS Start Up Kit – Automation.

http://www.waters.com/automate


2 GlycoWorks RapiFluor-MS N-Glycan Kit – Automation

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.

Figure 3. Workflow for the rapid preparation of N-glycans using the RapiFluor-MS N-Glycan Kit.

RapiFluor-MS

O NH

ON

O

NHN

N

O

O

Rapid Fluorescence

MS

NHS Carbamate RapidTagging Group

QuinolinylFluorophore

Tertiary AmineCharge Tag

AccQ·Fluor

RapidFluorescence

RapiFluor-MS Reagent

Streamlined protocol for a simple and fast sample preparation that minimizes sample handling and loss

5 minutes

10 minutes

LabeledN-Glycans

LabeledN-Glycans

+ ReagentsReaction

By-Products

Released N-Glycans + Protein

Glycoprotein

STEP 1: Deglycosylation

STEP 2: Labeling

STEP 3: Clean-up

LC-FLR LC-FLR-MS

Analysis

< 15 minutes

O NH

ON

O

NHN

N

O

OOH

N

O

O

+

3GlycoWorks RapiFluor-MS N-Glycan Kit – Automation

Table 1. Kit ContentsPerform kit inventory on arrival, do not use this product if its packaging is opened, damaged, items are missing, or beyond expiry date, if applicable. Replacement kit or parts can be ordered by contacting your local sales representative. Please include product part number and lot number for each item requested.

Kit format: 2 sets of 48 reactions can be combined to process 96 samples, if desired.Kit Component Purpose Qty. in KitGlycoWorks Deglycosylation Module 1 box containingIntact 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 – 2 x 48 1 box containing

GlycoWorks Rapid PNGase FGlycoWorks Rapid PNGase F (Kit Component) = 0.080 mL of PNGase F enzyme for use with rapid deglycosylation procedures. Recombinant source.

2 vials

GlycoWorks Rapid BufferGlycoWorks Rapid Buffer (1.50 mL of 250 mM HEPES pH 7.9) For use with deglycosylation/labeling.

2 vials

RapiGest™ SFAn enzyme-friendly surfactant used to denature the glycoproteins and to facilitate the deglycosylation reaction. Each set of 48 reactions requires one 20 mg vial.

2 vials

GlycoWorks RapiFluor-MS Labeling Module – Automation 1 box containingGlycoWorks RapiFluor-MS Reagent Powder

55 mg/vial of RapiFluor-MS reagent is included for labeling 48 samples.

2 vials

GlycoWorks Reagent Solvent Anhydrous DMF

1 mL ampoule of anhydrous dimethylformamide (DMF) solvent is used for solubilization of the RapiFluor-MS reagent.

2 ampoules

GlycoWorks RFMS Clean-up Module(Includes SPE plate and reagents in 2 separate boxes) 1 box containing

GlycoWorks HILIC µElution Plate

For removing potential interferences like labeling reaction by-products 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 Reagents 1 box containingGlycoWorks Automation SPE Elution Buffer

12 mL/vial 200 mM ammonium acetate in 5% ACN, eluent that is used for optimum glycan recovery from the HILIC SPE plate.

2 vials

GlycoWorks Automation Sample Diluent

22 mL/vial DMF solvent provided to dilute SPE eluate in preparation for HILIC chromatography.

2 vials

GlycoWorks Automation SPE Equilibration Solvent

18 mL/vial 15:85 water/acetonitrile for equilibration of the SPE plate. 2 vials

GlycoWorks Automation Wash Solvent

70 mL/vial 1:9:90 (v/v/v) formic acid/water/acetonitrile for washing of the SPE plate.

2 vials

Continued →

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.

http://www.waters.com/sds



Table 1. Kit Contents – continued

Kit format: 2 sets of 48 reactions can be combined to process 96 samples if desired.Kit Component Purpose Qty. in KitGlycoWorks Sample Collection Module – Automation 1 box containing1 mL Round Collection Plate (96-well)

Recommended for the collection of sample eluate during the SPE step. Second plate recommended for dilution step.

2 plates

200 µL PCR Plate (96-well) Recommended for the Deglycosylation reactions. 1 plate

Pre-Slit Cap MatCap mat recommended for use with the 1ml collection plate for direct injection after labelling and SPE.

1 mat

Waste TrayWaste tray provided to capture DMF waste in SPE manifold thereby preventing excessive wear on pump.

1 tray

Optional PN’s Included in Starter Kits 1 box containing

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 LC-MS grade bottle of water to get the recommended concentration of 5050 mM ammonium formate, pH 4.4.

1 vial

ACQUITY® UPLC® Glycan BEH Amide Column (available in all Starter Kits)

130Å, 1.7 µm, 2.1 x 150 mm 1 column

II. STORAGE AND STABILITY

GlycoWorks RapiFluor-MS N-Glycan Kit – Automation Components Storage Checklist

Standard/ReagentInitial Storage

After Reconstitution Source

Intact mAb Check Standard (unlabeled)

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)

RapiFluor-MS Intact mAb Mass Check Standard (deglycosylated, labeled, and purified)

Freeze on arrival (-20 °C)

4–10 °C for 1 week or sub-aliquot and freeze (-80 °C) for 3 months. AVOID freeze/thaw cycles.

Product COA

GlycoWorks Rapid PNGase F and GlycoWorks Rapid Buffer

Refrigerate (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 – Automated

Care and Use Manual (715005359)

RapiGest SFRoom 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 SF Surfactant Care and Use Manual

(715000122)

Continued →

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.

http://www.waters.com/waters/support.htm?lid=134695464&type=USRM


http://www.waters.com/sds


GlycoWorks RapiFluor-MS N-Glycan Kit – Automation Components Storage Checklist – continued

Standard/ReagentInitial Storage

After Reconstitution Source

RapiFluor-MS LabelRoom 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 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 SPE Reagents – Automation [15:85 water: acetonitrile, 1:9:90 (v/v/v) formic acid:water: acetonitrile and 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 , which is typically stable for about one month from initial opening. Once SPE diluent is opened, use it right away to avoid ACN evaporation to minimize chromatography solvent effects. All other reagents can be stored for up to 28 days once opened.



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.



Ammonium Formate Solution (Mobile Phase A Concentrate)

(Starter Kits or optional p/n)

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 temperature 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

(Starter Kits or optional p/n)

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.

Care and Use Manual (720003042EN)

Part numbers of available accessories can be found at the end of this document.



III. USING THE GLYCOWORKS RapiFLUOR-MS N-GLYCAN KIT – AUTOMATIONThe GlycoWorks RapiFluor-MS N-Glycan Kit – Automation is designed to provide scientists with all materials needed to successfully perform fast N-glycan deglycosylation, rapid fluorescent labeling, and glycan extraction with automation platforms.

Table 2. Checklist of Additional Materials for Sample Preparation Needed Before You BeginMaterial Description*

Recommended Suppliers

Formic AcidLC-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).

Part numbers of available accessories can be found at the end of this document.

GETTING STARTEDEnsure that the appropriate hardware is installed on the automated pipetting platform. Additionally, ensure that the software (and software options, if required) are installed on the automated pipetting platform as well as the installation of carriers and labware on the worktables (see Figure 4). As an example of workstation requirements for full automation, Tecans® Freedom EVO® 100/4 is used in this document as that was the original platform that the kit was verified on for full automation within this care and use manual.

It is recommended to follow the manufacturer's instructions in relation to system maintenance or if there are any questions related to specific workstation requirements, software, or scripting specific to your platform.

STEP 1. SETUP OF AUTOMATED PIPETTING PLATFORM FOR GLYCAN ANALYSIS

Example hardware requirementsEnsure that the following Tecan hardware is installed on the Tecan automated pipetting platform. If using other platforms, these can be examples of what is needed to run full automation of this workflow.

Description ExampleAutomated pipetting platform

Tecan Freedom EVO 100/4

Pipetting arm LiHa*

Gripper arm RoMa

Plate shaker Teleshake or equivalent

Heat block**INHECO CPAC or equivalent

Vacuum manifold and pump Te VacS or equivalent

*The LiHa arm used for development of the script contains four standard tips, therefore the script may need to be adjusted to suit different tip options.**Please take caution when operating heat blocks due to high temperatures.

Tecan Software Requirements and OptionsEnsure that the following software (and software options, if required) are installed on the Tecan automated pipetting platform PC.

Description Part Numbers

Pipetting softwareFreedom EVOware® (2.6 SP2 or equivalent) (p/n: 10615154)

Pipetting scriptRapiFluor-MS Pipetting Script (p/n: 715005361)

File converterMassLynx® V4.1 Tecan File Converter (p/n: 667004831) (optional for Sample Tracking if using MassLynx)

Export files

Microsoft® Excel with files 715005361 Import.xlsx template and 715005361 Import.csv (optional for Sample Concentration Normalization Feature) Import.gwl

Tecan Worktable LayoutEnsure that the following carriers and labware are installed on the worktable as shown in Figure 4.

A1

Dilution Plate

Carrier additivetrough (3 x 100 mL)

Waste tray

A1

Reaction Plate

Teleshake

Teleshake Base Plate

A1

Extraction Plate

Was

te

A1

Te-VacS

HeatBlock

PosID

1 mLcollectionplate sits

underreaction

plate

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Rack strip carrier 16 x 1.5 mLEppendorf tubes


Figure 4. Initial worktable layout for the GlycoWorks RapiFluor-MS N-Glycan Kit.

http://www.waters.com/waters/partDetail.htm?partNumber=186007081


Description Part Numbers

16-Position rack stripRack strip 16 position 16 x 1.5 mL Eppendorf (x7) Samples Tube Rack (p/n: 10613035)

Carrier 2MTP 1 Teleshake (x1) (Tecan) (p/n: 30029147)

Additive trough holder

Carrier Additive Trough Holder (x3) Reagent Trough Holders (p/n: 10613020)

Vacuum manifold/plate extraction manifold

Te-VacS vacuum manifold (x1) SPE plate extraction manifold

Tubes Microfuge Eppendorf tubes (1.5 mL)

Collection plates

Collection Plates (dilution plate and collection plate): Waters 96 -well 1 mL (included in GlycoWorks Sample Collection Module)

Reaction platesReaction Plates: 200 µL PCR Plate (included in GlycoWorks Sample Collection Module)

µElution extraction plates

Waters HILIC µElution Extraction Plates (included in GlycoWorks Sample Collection Module)

Waste tray Glycoworks Waste Tray (p/n: 600001282)

STEP 2. PREPARATION OF THE TECAN FREEDOM EVO 100/4

1. Log in to EVOware using the required username and password to load the startup screen.

2. Ensure the system has been primed with the correct system liquid before running.

Sample Tracking1. Samples are placed in the 16-position rack strip and

transferred to a 96-well reaction plate.

2. If a partial plate is used, sample 1 is transferred into the first well position selected by the analyst in the Tecan script dialog box. Subsequent samples follow on from this position.

3. The diluted sample is then transferred onto the SPE plate in the same well positions as the reaction plate.

4. Samples will then be eluted into the collection plate. Figure 5 shows a schematic of how samples are transferred when running p/n: 715005361.

Figure 5: Schematic of sample tracking.

1 2 3 4 5 6

Sample 1

Sample 16 Sample 96Sample 96

Sample 1

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Sample 96

Sample 1

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Sample Rack Reaction Plate SPE/Collection Plate

Barcoding and use of MassLynx File ConvertorThe Tecan EVOware script for the GlycoWorks RapiFluor-MS N-Glycan Kit - Automation has the capability to perform sample barcode scanning. Scanned sample information (e.g., sample I.D., sequence) may be recorded to a .csv file by the Tecan File Converter Software and this file may be later imported as a MassLynx V4.1 ready sample list.

When using this feature, take care to ensure the barcodes are as straight as possible; if a barcode is positioned diagonally on the tube it may not be read. Accepted barcode types currently setup on the RapiFluor-MS script are as follows:

Codabar 2 of 5 Interleaved Code 39 full ASCIICode 128 Code 39 Code 93

Ensure the Tecan File Converter Software is running.

Note: If the use of barcoded labels and sample tracking is not required by the user, place the samples in unbarcoded Eppendorf tubes at the start of the script. The Tecan will alarm during the barcode reading script step and requires the manual entry of sample numbers to progress to the next step. Alternatively, the user may create a modified script which has removed the barcode reading step altogether.

STEP 3. PREPARING TO RUN THE SCRIPT

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 48 samples:

50 mL of 18.2 MΩ water

25 mL of acetonitrile

Glycoprotein sample—— 60 µL of glycoprotein sample solution is recommended

for use with this protocol. For the optional sample concentration normalization feature, the 60 µL must be accurately dispensed into the eppendorf tube.

—— The recommended glycoprotein concentration is 1.5 mg/mL. However the script has an optional concentration normalization feature to dilute all samples between 1.5 mg/mL – 20mg/mL down to 1.5 mg/mL.



Intact mAb mass check standard (1 mg/vial)—— Use of a control standard is highly recommended. Intact

mAb Mass Check Standard is provided.

1. To achieve a recommended concentration of 1.5 mg/mL reconstitute 1 vial of mAb mass check standard in 667 µL of 18.2 MΩ Water.

2. Vortex to mix.

The user will need to prepare one vial of each reagent for the generation of 48 samples:

Buffered RapiGest SF solution (20 mg per vial)1. Dissolve the contents of 1 vial of Rapigest SF in 400 µL

of 5x Glycoworks Rapid Buffer and 270 µl 18.2 MΩ water.

2. Vortex to mix.

Buffered PNGase F solution (80µl per vial)1. Add 586.7 µL of 5x Glycoworks Rapid Buffer to a vial of

PNGase F.

2. Vortex to mix.

RapiFluor MS Reagent solution (55 mg per vial)Prepare reagent solution by dissolving one vial of 55 mg of the GlycoWorks RapiFluor-MS Reagent Powder in 669.4 µL of the provided GlycoWorks Reagent Solvent Anhydrous DMF. Aspirate and dispense the solution 5–10 times to ensure the reagent is dissolved. Alternatively, cap and briefly vortex the contents of the reagent vial.

Reagent PreparationEnsure all reagents are prepared and ready for use according to the Care and Use Manual included in kit.

Ensure all reagents are placed in the correct troughs (as shown in Figure 6) and that there are sufficient volumes present for the amount of samples to be analysed (Table 3).

When placing the RapiGest SF Solution in the designated Eppendorf Tube, avoid the formation of bubbles in the tube as this may affect the automated pipetting.

Table 3. Reagent and Solvent Volumes Guide

Reagent/SolventSample

Quantity - 48Sample

Quantity - 96Labware

RapiGest SF Surfactant 670 µL 1340 µL 1.5 mL Eppendorf tube

PNGase F 666.7 µL 1333.4 µL 1.5 mL Eppendorf tube

RapiFluor-MS Label 669.4 µL 1338.8 µL 1.5 mL Eppendorf tube

15:85 water: acetonitrile 18 mL 36 mL 100 mL trough1:9:90 (v/v/v) formic acid:water:acetonitrile 70 mL 140 mL 100 mL troughSPE elution buffer 12 mL 24 mL 100 mL trough32:68 DMF:ACN 22 mL 44 mL 100 mL trough18.2 MΩ water 50 mL 100 mL 100 mL troughAcetonitrile (HPLC grade) 25 mL 50 mL 100 mL trough

32:68DMF:acetonitrile

1:9:90 Formic acid:water:

acetonitrile

Waste 15:85 Water:acetonitrile

SPE elution buffer

1:9:90 Formic acid:water:

acetonitrile

18:2 MΩ:WaterAcetonitrile

Back

Front

15 16 17 18 19

RapiGest SF

PNGase F

RapiFluor-MS

Figure 6: Solvent/reagent worktable layout for a 96 sample run.

Note: When preparing 48 samples the second (back) 1:9:90 formic acid/water/acetonitrile trough is not required.


Sample Dispensing and BarcodingIf tracking the sample sequence using barcodes:

1. Check to ensure that all the samples that are to be analysed have a barcode on them, and confirm that the MassLynx V4.1 Tecan File Converter Software is running.

2. Carefully pipette the samples to be tested into the bottom of each Eppendorf Tube, an aliquot of 60 µL is added. This 60-µL volume must be accurate for use with the sample concentration normalization feature.

Note: More than 60 µL can be added to the Eppendorf tube if the normalization feature is not being used.

3. Place the sample tubes into the sample racks so the barcodes are visible through the slots on the right hand side. Figure 7 shows the barcode position on the sample tube rack.

Sample Concentration Normalization (Optional)If sample concentration normalization is to be performed with the run, ensure that all glycoprotein sample concentrations are updated into the "concentrated" column of the 715005361 Import.xlsx template as per Figure 8.

The number of samples to be tested should also be updated as the script will run to the last line available in the template.

The file should then be saved only as a .csv file under the name 715005361 Import.csv. The script will then look for this .csv file name and will convert the script into a .gwl file to carry out the normalization step.

Figure 7. Barcode position on sample tube rack.

STEP 4. EXECUTING THE SCRIPT FOR AUTOMATED SAMPLE PREPARATION

Startup1. Install script provided on CD. Ensure the taught settings

for the worktable are correct before proceeding. Failure to do so may result in a collision.

2. In the EVOware startup screen select run an existing script and click on the current GlycoWorks RapiFluor-MS script in the script selection screen.

3. Ensure that the Run full script box is checked and click the green arrow RUN button.

4. A number of prompts will appear during the script run. An initial prompt to carry out systems checks will appear as per Figure 6. Once the prompt has been followed, click on the Ok button to continue.

Figure 8. Normalization worksheet template example.

5. The script will then display three user input prompts as per Figure 10. The user must input the file name to be given to the run [e.g., 161220_0001_Glycan (if using the Masslynx Tecan file converter, it is important to include the template name "Glycan" at the end of the file name to ensure all data will be exported)], how many samples will be prepared (e.g., 48 or 96), and which well to start at. Click Ok on each prompt once the relevant information has been entered.

Figure 9. Initial user prompt.

Figure 10. User input prompts.



6. The next input prompt to display (see Figure 11) will ask the user if they would like to perform the normalization step by inputting Yes or No. The prompt will also remind the user to ensure the correct concentrations and number of samples has been updated into the normalization .csv file to be imported.

7. A user prompt will then appear when the collection plate is ready to be sealed with the pre slit cap mat provided (see Figure 12).

8. A final user prompt will then display to inform the user of the completion of the script (see Figure 13).

Figure 11. Normalization prompt.

GlycoWorks RapiFluor-MS Automation Script StepsFor reference please refer to the included Care and Use manual for script steps.

Last StepsOnce the Tecan script has completed, remove the sealed 96-well plate from the Tecan worktable and transfer it to the UPLC System for analysis.

Upon completion of the protocol ensure that any waste generated during the run is disposed of in the appropriate waste stream.

Note: If not running the full automated script the manual settings for the Boekel heat block and the bench top vacuum can be found in the GlycoWorks RapiFluor-MS N-Glycan Kit – 96 Sample Care and Use Manual (p/n: 715004793EN) on www.waters.com.Figure 12. User prompt.

Figure 13. Final user prompt.


http://www.waters.com


Avoid SDS and Nucleophiles

Tris DTT Glycine HistidineTrTT is ycine tidine

Ammonium Mercaptoethanol

— 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.

— This protocol is optimized and 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 labelling reagent, which will potentially result in low yield or so-called over labellng artifacts.

— 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.

— 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.

— Bring PNGase F up to room temperature and centrifuge before use.

O NH

ON

O

NHN

N

O

OOH

N

O

O

+

Rapid Fluorescence

MS

— RapiFluor-MS is purified as a 1:1 complex with NHS. The formula weight for the reagent as provided is 542.41 g/mol.

— 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.

— Glycans are released from glycoproteins as glycosylamines, an important fact for an analyst to consider when using this labeling chemistry.

RapiFluor-MS Reagent Powder.

GlycoWorks RapiFluor-MS Script Steps

Test section Step description comment lines

Normalization Normalization step - Import and execute worklist.

Deglycosylation1 Add 10 µL of Glycoprotein sample

into the reaction plate.2 Add 10 µL of RapiGest SF to reaction

plate and mix for 1 minute.3 Move the reaction plate to the heat

block at 90 °C for 3 minutes. 4 Move reaction plate back to

Teleshake to cool for 3 minutes. 5 Add 10 µL of PNGase F to reaction

plate and mix for 1 minute. 6 Move reaction plate to heat block

at 55 °C for 5 minutes. 7 Move reaction plate back to

Teleshake to cool for 3 minutes.

Labeling8 Add 10 µL of RapiFluor MS to reaction

plate, mix for 5 minute reaction.9 After 5 minutes dilute with

360 µL of acetonitrile.

Overview of Workflow:Below is a summary of the workflow that the script will be running for reference and tips and tricks of each step incorporated from the original manual method for reference.



A1

Dilution Plate



Waste tray

A1

Reaction Plate

Teleshake


A1

Extraction Plate

Was

te 1 mL Collection Plate

A1

Te-VacS

HeatBlock

PosID

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30


Barcode scanning ofsamples and normalization.

Transfer of sample anddilution with acetonitrile.

Addition of 10 µL Glycoprotein, RapiGest,PNGase F and RapiFluor MS.

Aspiration ofGlycoprotein samples.

Perform denaturation at 90 °Cand deglycosylation at 50 °C.

Conditioning (18.2 MΩ water),equilibration (15:85

water:acetonitrile), loading ofsamples and wash step (1:9:90formic acid/water/acetonitrile).

Elution of glycans (SPEelution buffer) into collection

plate then eluate dilution(32:68 DMF:ACN).

A1

Dilution Plate


Waste tray

A1

Reaction Plate

Teleshake


A1

Extraction Plate

Was

te 1 mL Collection Plate

A1

Te-VacS

HeatBlock

PosID

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30



1

2

3

6

9

4

7

5

8

10

11

13

14

13

15

16

Figure 14. Schematic representation of the Tecan Freedom EVO 100/4 bed layout for glycan analysis.

SPE cleanup10 Condition extraction plate with

200 µL of 18.2 MΩ water. 11 Equilibrate extraction plate with

200 µL of 15:85 water/acetonitrile. 12 Load diluted samples (~400 µL)

to extraction plate. 13 Wash extraction plate with two

600 µL volumes of 1:9:90 Formic acid/water/acetonitrile.

14 Transfer extraction plate over 1 mL collection plate and elute sample using five 18 µL volumes of SPE elution buffer.

Final sample preparation15 Dilute 90 µL of eluate with 310 µL of

32:68 DMF/ACN. 16 Seal the collection plate for analysis

using pre slit cap mat.

— 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. It is especially important to ensure this heat transfer with automation. It is recommended to calibrate your heating device before use for the hardware you are using.


STEP 5. RECOMMENDED ANALYTICAL APPROACH FOR GLYCOWORKS RapiFLUOR-MS LABELED GLYCAN ANALYSIS

Before You Get Started: Glycan BEH Amide Column EquilibrationWhen 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 (Method used with the RapiFluor-MS Library)Column: ACQUITY UPLC Glycan BEH Amide, 130Å, 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 mobile phase concentrate: Ammonium formate (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 Flow rate (min) (mL/min) %A %B Curve 0.0 0.4 25 75 6 35.0 0.4 46 54 6 36.5 0.2 100 0 6 39.5 0.2 100 0 6 43.1 0.2 25 75 6 47.6 0.4 25 75 6 55.0 0.4 25 75 6

FLR wavelengths: EX 265/EM 425 nm

FLR sampling rate: 10 Hz

— 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.

Mobile Phase Recommendations

It is critical to use LC-MS-grade solvents and buffers when performing LC-MS glycan analysis.

Seal wash and LC 70% LC-MS-grade ACN autosampler wash: 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.

Figure 15. 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.

+

+

H2O

+ + CO2

Mass shift from glycosylamine

+C17H2ON4O2 (ΔMi = 312.1586 Da/ΔMAvg = 312.3663 Da)

Mass shift from glycans with free reducing end

+C17H21N5O1 (ΔMi = 311.1746 Da/ΔMAvg = 311.3815 Da)







mAb N-Glycan Profiling MethodLC system: ACQUITY UPLC H-Class Bio

Sample temp.: 10 °C

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, 130Å, 1.7 µm, 2.1 x 150 mm (p/n: 186004742)

Fluorescence detection: Ex 265 nm/Em 425 nm, 2 Hz

Mobile phase A: 50 mM ammonium formate, pH 4.4 (LC-MS grade; from a 100x concentrate) (p/n: 186007081)

Mobile phase B: ACN (LC-MS grade)

Please see Application Note 720005516EN.

Gradient: Time Flow rate (min) (mL/min) %A %B Curve 0.00 0.5 20 80 3.00 0.5 27 73 6 35.0 0.5 37 63 6 36.5 0.2 100 0 6 39.5 0.2 100 0 6 43.1 0.2 20 80 6 47.6 0.5 20 80 6 55.0 0.5 20 80 6

Autosampler Settings for UNIFI and EmpowerThe Tecan script for the Glycoworks RapiFluor-MS N-Glycan Kit- Automated uses ANSI 96-well, 1-mL plates for the final analytical sample.

Configure the UPLC autosampler bed using Empower,® MassLynx or UNIFI® to inject from ANSI 96-well 1-mL plates (example: Empower Autosampler Configuration Table 4).

UNIFI: Depending on the version of UNIFI, users will find the plate is already configured. Search for “GlycoWorks Plate” under Consumables.

Figure 16. Autosampler configuration for Empower.



http://www.waters.com/waters/library.htm?cid=511436&lid=134865659&locale=en_US


Time9.00 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 1

0e4

0.000

200000.016

400000.031

600000.063

800000.063

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 1

0e4

0.000

200000.016

400000.031

600000.063

800000.063

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 1

0e4

0.000

200000.016

400000.031

600000.063

800000.063

A2

FA2

A2G1FA1G1

A2G1

FA2G1*

FA2G2

FA2G2Ga1

FA2G2Sg1FA2G2Ga2

800000.063

9.0010.0011.0012.0013.0014.0015.0016.0017.0018.0019.0020.0021.0022.0023.0024.00

EU

x 1

0e4

400000.031

600000.063

A2

FA2

A2G1FA1G1

A2G1

FA2G1*

FA2G2

FA2G2Ga1

FA2G2Sg1FA2G2Ga2

Zoomed View

m/z m/z

Glycan

* First peak is F(6)A2[6]G(4)1, the second is F(6)A2[3]G(4)1.

RapiFluor-MS Labeled Glycan

CompositionMi (Da) 1+ 2+ 3+ Mavg (Da) 1+ 2+ 3+

A2 C67H105O37N9 1627.6611 1628.6684 814.8378 543.5610 1628.5887 1629.596 815.30165 543.8702

FA2 C73H115O41N9 1773.7190 1774.7263 887.8668 592.2469 1774.7299 1775.7372 888.37225 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

For more detailed characterization of the RapiFluor-MS Intact mAb Standard (deglycosylated, labeled, and purified) with a RFMS comparison, see “Robustness of RapiFluor-MS N-Glycan Sample Preparations and Glycan BEH Amide HILIC Chromatographic Separations” (p/n: 720005370).

Representative HILIC-FLR-MS data for RapiFluor-MS Intact mAb Standard (p/n: 186008843) (deglycosylated, labeled, and purified) using a Waters SYNAPT G2-S HDMS.

http://www.waters.com/webassets/cms/library/docs/720005370en.pdf



IV. ORDERING INFORMATION (For the most up to date product listing, click here)

Sample Preparation Complete KitsDescription P/N

GlycoWorks RapiFluor-MS N-Glycan Base Script Starter Kit - Automation

GlycoWorks RapiFluor-MS Sample Prep Script Parameters Document; Intact mAb Mass Check Standard (unlabeled); RapiFluor-MS Intact mAb Mass Check Standard (deglycosylated, labeled, and purified); GlycoWorks Rapid Deglycosylation Kit – 2 x 48; GlycoWorks RapiFluor-MS Labeling Module – Automation; Glycoworks HILIC µElution Plate; GlycoWorks SPE Reagents – Automation; GlycoWorks Sample Collection Module – Automation; ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7 µm, 2.1 x 150 mm Column; Mobile phase concentrate: ammonium formate.

176004150

GlycoWorks RapiFluor-MS N-Glycan Script Starter Kit – Automation

GlycoWorks Automation Script Pack-CD; Intact mAb Mass Check Standard (unlabeled); RapiFluor-MS Intact mAb Mass Check Standard (deglycosylated, labeled, and purified); GlycoWorks Rapid Deglycosylation Kit – 2 x 48; GlycoWorks RapiFluor-MS Labeling Module – Automation; Glycoworks HILIC µElution Plate; GlycoWorks SPE Reagents – Automation; GlycoWorks Sample Collection Module – Automation; ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7 µm, 2.1 x 150 mm Column; Mobile phase concentrate: ammonium formate.

176004151

GlycoWorks RapiFluor-MS N-Glycan Starter Kit – Automation

Intact mAb Mass Check Standard (unlabeled); RapiFluor-MS Intact mAb Mass Check Standard (deglycosylated, labeled, and purified); GlycoWorks Rapid Deglycosylation Kit – 2 x 48; GlycoWorks RapiFluor-MS Labeling Module – Automation; GlycoWorks HILIC µElution Plate; GlycoWorks SPE Reagents – Automation; GlycoWorks Sample Collection Module – Automation; ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7 µm, 2.1 x 150 mm Column; Mobile phase concentrate: ammonium formate.

176004152

GlycoWorks RapiFluor-MS N-Glycan Kit - Automation

GlycoWorks Rapid Deglycosylation Kit – 2 x 48, GlycoWorks RapiFluor-MS Labeling Module – Automation, GlycoWorks HILIC µElution Plate, GlycoWorks SPE Reagents – Automation and GlycoWorks Sample Collection Module – Automation.

176004153

GlycoWorks RapiFluor-MS N-Glycan Basic Kit - Automation

GlycoWorks Rapid Deglycosylation Kit – 2 x 48, GlycoWorks RapiFluor-MS Labeling Module – Automation, GlycoWorks HILIC µElution Plate and GlycoWorks SPE Reagents – Automation.

176004154

ColumnsDescription Qty. P/NACQUITY UPLC Glycan BEH Amide, 130Å, 1.7 µm, 2.1 x 50 mm Column 1 186004740ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7 µm, 2.1 x 100 mm Column 1 186004741ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7 µm, 2.1 x 150 mm Column 1 186004742XBridge Glycan BEH Amide XP, 130Å, 2.5 µm, 3.0 x 75 mm Column 1 186008039XBridge® Glycan BEH Amide XP, 130Å, 2.5 µm, 3.0 x 150 mm Column 1 186008040ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 µm 2.1 x 50 mm with Std 1 176003700ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 µm 2.1 x 100 mm with Std 1 176003701ACQUITY UPLC Glycoprotein BEH Amide, 300Å , 1.7 µm 2.1 x 150 mm with Std 1 176003702Glycoprotein Performance Test Standard 1 186008010







V. RESOURCES AND REFERENCES

DescriptionLiterature

Code*Application NotebookGlycan Application Notebook 720005532EN

Video

Training Video www.waters.com/RapiFluorMS

Care and Use Manuals

RapiFluor-MS Dextran Ladder Calibration 720005348EN

RapiFluor-MS Glycan Performance Test Standard 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

Standards and AccessoriesDescription Qty. P/NIntact mAb Mass Check Standard* 1 mg/vial 186006552GlycoWorks RapiFluor-MS Dextran Calibration Ladder 50 µg/vial 186007982RapiFluor-MS Intact mAb Standard 400 pmol/vial 186008843

GlycoWorks RapiFluor-MS Glycan Performance Test Standard 400 pmol/vial 186007983

GlycoWorks RapiFluor-MS High Mannose Standard 1000 pmol/vial 186008317RapiFluor-MS Quantitative Glycan Standard 100 pmol/vial 186008791RapiFluor-MS Sialylated Glycan Performance Test Standard 400 pmol/vial 186008660GlycoWorks Rapid Deglycosylation Kit – 2 x 48 2 x 48 reactions 186004579RapiGest SF 2 x 20 mg/vials 186008746GlycoWorks Rapid Buffer 5 mL/vial 186008100GlycoWorks RapiFluor-MS Labeling Module – Automation 1 box 186008822Glycoworks HILIC µElution Plate 1 plate 186002780GlycoWorks SPE Reagents – Automation 1 kit 186008747GlycoWorks Sample Collection Module – Automation 1 box 186008823Ammonium Formate Solution – Glycan Analysis (Mobile Phase Concentrate) 5050 mM/vial 186007081Certified Container Kit Kit contains: (4) 1 Liter bottles, (3) 500 mL bottles, and 1 Cap Kit

1 186007088

*Control Standard included in kit.









DescriptionLiterature

Code*

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

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


VI. COMMON GLYCAN NAMES, MASSES, AND CHARGE STATES

Waters RapiFluor-MS Glycan GU Scientific Library: www.waters.com/glycanUNIFIGlycan Oxford Notation

GlycanRapiFluor-MS Labeled Glycan Composition

Mi (Da) 2+ 3+ Mavg (Da) 2+ 3+

M5 Man5 C63H99O37N7 1545 .608 7 73 .8113 516 . 2099 1546 .49 7 74 . 25 516 .5A2 G0 C67H105O37N9 1627.6611 814 .8378 543 .561 1628 .59 815 .3 543 .87M6 Man6 C69H109O42N7 1707.6609 854 .837 7 570. 2276 1708 .63 855 .32 570.55

FA1G1 G1F-GN C71H112O41N8 1732 .6925 867.3535 578 .5714 1733 .68 867.85 578 .9

FA2 G0F C73H115O41N9 17 73 .7 19 887.8668 592 . 2469 17 74 .73 888 .37 592 .58A2G1 G1 C73H115O42N9 1789.7 14 895 .8643 597.5786 1790.73 896 .37 597.92FA2G1 G1F C79H125O46N9 1935 .7 719 968 .8932 646 . 2646 1936 .87 969.44 646 .63A2G2 G2 C79H125O47N9 1951 .7668 976 .8907 651 .5962 1952 .87 97 7.44 651 .96FA2B G0F+GN C81H128O46N10 1976 .7984 989.4065 659.9401 197 7.92 989.97 660.31FA2G2 G2F C85H135O51N9 2097.8247 1049.9196 700. 2822 2099.01 1050.51 700.68FA2BG1 G1F+GN C87H138O51N10 2138 .8512 1070.4329 713 .957 7 2140.06 1071 .04 714 .36FA2G1S1 G1F+NANA C90H142O54N10 2226 .8673 1114 .4409 743 . 2964 2228 .13 1115 .07 743 .72FA2G2Ga1 G2FGal1 C91H145O56N9 2259.87 75 1130.946 754 . 2998 2261 .15 1131 .58 754 .72FA2BG2 G2F+GN C93H148O56N10 2300.9041 1151 .4593 767.9753 2302 . 2 1152 .11 768 .41FA2G2S1 G2F+NANA C96H152O59N10 2388 .9201 1195 .4673 797.314 2390. 27 1196 .14 797.76FA2G2Sg1 G2F+NGNA C96H152O60N10 2404 .915 1203 .4648 802 .6456 2406 . 27 1204 .14 803 .1FA2G2Ga2 G2FGal2 C97H155O61N9 2421 .9303 1211 .9724 808 .3174 2423 . 29 1212 .65 808 .7 7FA2BG2S1 G2F+GN+NANA C104H165O64N11 2591 .9995 1297.007 865 .0071 2593 .46 1297.74 865 .49FA2G2S2 G2F+2NANA C107H169O67N11 2680.015 1341 .0148 894 .3456 2681 .52 1341 .7 7 894 .85FA2BG2S2 G2F+GN+2NANA C115H182O72N12 2883 .0949 1442 .5547 962 .0389 2884 .7 1 1443 .36 962 .58

Mass Spectrometry Analysis ConsiderationsBecause the RapiFluor-MS Label has high proton affinity, derivatized glycans preferentially adopt high charge states during positive ion mode electrospray ionization. The 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.


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.

©2017 Waters Corporation. Printed in the U.S.A. ALL RIGHTS RESERVED. THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER.

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Documents

GlycoWorks RapiFluor-MS N-Glycan Kit – Automation