glykoprep - plus rapid n-glycan sample preparation with ......for this protocol you should be...

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GlykoPrep -plus Rapid N-Glycan Sample Preparation with APTS Automated on AssayMAP Technology®

An automated, highly robust, high-throughput process for enzymatic deglycosylation, fluorescent labeling with APTS and cleanup of excess dye foranalysis by CE and other methods.

� Non-selective, rapid release and recovery of N-glycans from up to 96 glycoprotein samples at a time on the Agilent AssayMAP Bravo LiquidHandling Workstation (AssayMAP Bravo)

� Minimal hands-on time

� Optimized reaction conditions help to preserve labile glycans, such as sialic acid

� Non-selective chemistry for stoichiometric labeling of glycans, independent of structure

� Complete labeling in 1 hour using Rapid-Reductive-Amination™

� Purified APTS-labeled N-glycans are eluted in water, ready for analysis

Product Code: GPPNG-APTS

NOTICE: ProZyme was purchased by Agilent in July 2018. Documents for products and product lots manufactured before August 2019 will contain references to ProZyme. For more information about these products and support, go to: www.agilent.com/en/contact-us.

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TABLE OF CONTENTS

Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

How to Use This Booklet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

GPPNG-APTS Kit Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Storage Requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Additional Required Reagents/Equipment. . . . . . . . . . . . . . . . . . . . . . . . 5Optional Reagents and Supplies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Safety and Handling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Using the Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6GlycoProtein Sample Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Using the Reagent Volume Calculator.. . . . . . . . . . . . . . . . . . . . . . . . . . 10GlykoPrep-plus Reagent Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . 11AssayMAP Bravo Processing Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Preparing for the GlykoPrep-plus Protocols. . . . . . . . . . . . . . . . . . . . . . 13

Glycoprotein Sample Plate Preparation. . . . . . . . . . . . . . . . . . . . . . 13Reagent Source Plate Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . 13Optional Labeling Source Plate Preparation. . . . . . . . . . . . . . . . . . . 14Preparation of the AssayMAP Bravo and Associated Equipment. . . 14

GlykoPrep-plus Protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Plate & Reagent Setup Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . 162 RX Cartridge Setup Protocol.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Immobilization & Digestion Protocol. . . . . . . . . . . . . . . . . . . . . . . 20Manual Processing - Drying the N-Glycans.. . . . . . . . . . . . . . . . . . . 234 Cleanup Cartridge Setup Protocol. . . . . . . . . . . . . . . . . . . . . . . . . 24Manual Processing - Labeling the N-Glycans. . . . . . . . . . . . . . . . . . 265 Labeling Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 Cleanup Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

QuickGuide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Analysis of Labeled Glycans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Tips & Hints. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Technical Assistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

License to Use.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Trademarks & Tradenames. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Product Use & Warranty. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Other ProZyme Products & Kits. . . . . . . . . . . . . . . . . . . . . . . . . 46

Ordering Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

This product is intended for in vitro research use only.

NOTE: The following suggestions and data are based on inform ation we believe to be reliable. They

are offered in good faith, but without guarantee, as conditions and methods of use of our products are

beyond our control. We recom m end that the prospective user determ ine the suitability of our materials

and suggestions before adopting them on a com m ercial scale. Suggestions for use of our products or

the inclusion of descriptive material from patents and the citation of specific patents in this publication

should not be understood as recom m ending the use of our products in violation of any patent or as

perm ission to license to use any patents of ProZym e, Inc.

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EQUIPMENT

The AssayMAP Bravo and associated equipment are available from Agilent Technologies.

Comments

AssayMAP Bravo Liquid Handling Workstation (AssayMAP Bravo, p/n G5542A) Bravo 96 AM Head (p/n G5498B#046)

Peristaltic Pump Module 2.0 (p/n G5498B#058) linked to 96AM Wash Station (p/nG5498B#057)

Peltier Thermal Station - with STC Controller (p/n G5498B#035)Cartridge Seating Station (aka Tips Loading Station)Orbital Shaking Station w/ Control Unit (p/n G5498B#033)96AM Cartridge & Tip Loading Station (p/n G5409-20025)Gripper upgrade (p/n 16545-101)Risers, 146 mm (p/n G5498B#055)Custom Plate Nest (p/n G5498B#017)PCR Plate Insert (p/n G5498B#013)Carboy to feed pump & collect waste ( 2 ea, G5550-17725)Lab tape (to tape the wash station, RX/CU Cartridge Racks and Receiver Plate to the

deck)

SOFTWARE

For this protocol you should be running the latest version of Agilent VWorks"GlykoPrep-plus Full App Installer with N-Glycan Sample Prep: RX digestion & APTS labeling v1.0 or newer

The latest version of this Instruction Manual may be found here:

www.prozyme.com/~prozyme/documents/GPPNG-APTS_Instruction_Manual.pdf

HOW TO USE THIS BOOKLET

We want successful results for our customers, so please read this entire bookletbefore starting the procedure, and follow the detailed instructions.

Once familiar with the AssayMAP Bravo and GlykoPrep-plus protocols, experiencedusers may wish to use the streamlined QuickGuide section beginning page 32.

AssayMAP Bravo must be installed, calibrated, and set upwith the GlykoPrep layout and recommended software.

Print the QuickGuide pages separately, using PortraitOrientation

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KIT CONTENTS

GPPNG-APTS GlykoPrep-plus Rapid N-Glycan Sample Preparation with APTS

GSP-RX GlykoPrep-plus Digestion ModuleWS0253 RX Cartridges (96)WS0256-GP Immobilization Reagent Set

WS0226-GP Denaturation Reagent (35 ml)WS0255-GP Blocking Reagent (14 ml)

WS0259-GP Digestion Reagent SetWS0229-GP Finishing Reagent (10 ml)WS0276-GP 25x Digestion Buffer (1.3 ml)WS0278-GP N-Glycanase (700 �l)

GS96-APTS GlykoPrep Rapid-Reductive-Amination APTS Labeling Module WS0299 APTS Solution (130 �l) WS0300 Reductant Solution (325 �l) WS0295 APTS Catalyst (325 �l) Aluminum Sealing Film

GSP-C2 GlykoPrep-plus APTS Cleanup ModuleWS0263 CU Cartridges (96)WS0301-GP 5x APTS Sample Load Buffer (48ml)Aluminum Sealing Film (2)

Labware Set from ProZyme (product code AM96-NG when purchased alone)

WS0304 1 ea, 12-Column, Reservoir Plate (5-pack)WS0305 4 ea, PCR PlateWS0306 2 ea, Square, Deep-Well PlateWS0307 1 ea, Pipette-Tip BoxWS0308 1 ea, U-Bottom Plate (10-pack)

Aluminum Sealing Film (2)

1 ea

1 ea

1 ea

1 ea

Confirm all materials are present before beginning.

Ships with Product Code GPPNG-APTS. These are thenames used throughout the protocols.

WARNING: The supplied protocols and applicationsupport a specific list of labware, which is included inthe purchase of the GPPNG-APTS Kit. If labware otherthan those listed here are to be used, the protocol andapp require editing. Using undefined or incorrectlyidentified labware on the AssayMAP Bravo deck cancause damage to the Bravo 96AM Head.

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Storage Requirements

This Kit is a mixed-temperature shipment (2–30�C). Upon arrival, storecomponents as indicated. For best results, equilibrate materials to ambienttemperature prior to use. The APTS Solution is light-sensitive and theReductant Solution and APTS Catalyst are hygroscopic; please store thesereagents at -20�C in their original packaging.

Additional Required Reagents/Equipment

Ultrapure, deionized water (~100 ml, Milli-Q or equivalent)®

100% Acetonitrile (~250 ml, HPLC-grade)Microplate-compatible, centrifugal evaporator (e.g., SpeedVac with plate®

rotor or similar)Fume hood (for Labeling procedure)VortexerNitrile GlovesCalibrated pipettes and disposable tips (P5/10, P200 and P1000)

Volumetric Pipettes (5-ml, 1 ea)Glass Graduated Cylinder (50-ml, 1 ea)Glass Bottle with Screw Cap (50-ml, 1 ea)

Optional Reagents and Supplies

Multichannel pipettors & disposable tips (P5/P10 and P200) (Gilson orequivalent, compatible with Gilson Diamond D200 pipette tips)®

SAFETY AND HANDLING

Some of the reagents in this Kit are hazardous. Please refer to the Safety DataSheets (SDS) included with the Kit or posted on ProZyme’s website under thecomponent name or Product Code.

http://www.prozyme.com

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General Laboratory Procedures

Use powder-free, nitrile gloves for all sample handling procedures. Ensure thatall glass, plasticware and solvents are free of glycosidases and environmentalcarbohydrates.

All procedures involving Labeling Reagents (APTS Solution, Reductant Solutionand APTS Catalyst) should be performed in a dry environment with dryglassware and plasticware, using appropriate personal safety protection,eyeglasses and nitrile gloves, and where appropriate, in a fume hood.

INTRODUCTION

The GlykoPrep-plus Sample Preparation Platform (GlykoPrep-plus) dramaticallystreamlines glycoanalysis by automating deglycosylation and separation ofN-glycans, complete fluorescent labeling and efficient cleanup to reduce excessreagent peaks using the Bravo 96AM Syringe Head on the AssayMAP Bravo.

GlykoPrep-plus is modular and can be integrated into any workflow, regardlessof throughput or sample type. In order to match any standard samplepreparation, Kit components may be made available individually; for moreinformation please contact us.

GlykoPrep-plus is built on AssayMAP technology, microchromatography in a96-well format using the Syringe Head on the Agilent AssayMAP Bravo tomove liquid through the Cartridges. The same procedures may be performedusing centrifugation (GlykoPrep, spin format). For more information aboutGlykoPrep, please visit our website:

http://www.prozyme.com/GlykoPrep/

USING THE KIT

GlykoPrep-plus Rapid N-Glycan Preparation with APTS combines the DigestionModule, the Rapid-Reduction-Amination APTS Labeling Module and theCleanup Module, which may be purchased individually. The Labeling andCleanup Modules may be purchased together as GPP-APTSCU.

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This GPPNG-APTS Instruction Manual provides instructions for use of the AssayMAPBravo for N-glycan processing using GlykoPrep-plus Rapid N-Glycan SamplePreparation with APTS Kit (GPPNG-APTS). It also provides additional backgroundinformation and explanations to frequently-asked questions regarding:

• Glycoprotien Sample Preparation• Using the Reagent Volume Calculator • AssayMAP Bravo Processing Time• GlykoPrep-plus Reagent Preparation

Glycoprotein Sample Preparation

This section discusses a number of considerations when preparing GlycoproteinSamples for processing on the AssayMAP Bravo.

� Optional Purification

The GPPNG-APTS Kit begins with purified Glycoprotein Samples. To process cellculture or other mixtures in a single workflow, a purification module, such asProtein A or Protein G, may be employed just prior to Protocol 1 on the AssayMAPBravo.

Glycoprotein Samples must not contain any particulates, as they will plug the topfrit, or sit on the top of the resin bed and impede the flow. Spin samples to removeparticulates before processing.

� Sample Quantities

The quantitative binding capacity for each Cartridge is:

RX Cartridge 50 �g of any standard proteinCU Cartridge 30 �g of N-glycans

The binding capacity for specific glycoproteins may needto be determined.

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Each Cartridge is capable of binding more target, but will do so with increasingbreakthrough, making the process non-quantitative. Less than the maximumquantity may be processed, for example, when the sample is available only inlimited amounts. The smallest effective amount of sample will depend on thesensitivity requirements of the analytical methods used and the specific application.

� Sample Concentrations

For standard processing, the Glycoprotein Sample concentration should be in therange of 1–5 mg/ml, and sufficient reagents have been provided to processindividual columns (8 samples) at a time. The GPPNG-APTS Kit can also be used formore dilute samples (down to 0.05 mg/ml); the effective minimum SampleConcentration will depend on the denaturation requirements for the specificglycoprotein (see below).

If quantitation is desired, pipetting less than 10 �l is notrecommended; pipetting smaller volumes introducesvariability, especially when samples are highlyconcentrated. If necessary, dilute the sample to withinthe 1-5 mg/ml range with Digestion Buffer beforebeginning.

NOTE: All Glycoprotein Samples will be loaded at thesame volume. To achieve uniform loading across all theCartridges, the Glycoprotein Samples must be adjusted tothe same concentration.

� Sample Denaturation

Prior to the enzyme digest, the Glycoprotein Samples are denatured by pre-mixingwith Denaturation Reagent to open up the protein structure and allow access to thedeglycosylation enzyme. The standard protocol employs a 5-minute, relativelygentle denaturation; the Denaturation Reagent is 6 M Guanidine. Start with a 1:1ratio of Glycoprotein Sample to Denaturation Reagent, and increase the ratio ofDenaturation Reagent to 9:1 to test the effects of increasing level of DenaturationReagent. Most glycoproteins can be adequately denatured under these conditions.

The amount of Denaturation Reagent to be added isdetermined by setting the Denaturation Factor in theReagent Volume Calculator (see instructions page 11).

Any custom denaturation may be performed prior toprocessing on the AssayMAP Bravo, as long as no SDS orother detergents are used.

In all cases, a minimum of 50% Denaturation Reagent should be added prior toloading onto the RX Cartridge. This assures that the Glycoprotein Sampleformulation matches the equilibration conditions of the RX Cartridge, and avoidsprecipitation of the protein, which will plug the Cartridge.

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� Deglycosylation Temperature

The assay has been optimized for a setting of 45�C. Note that this temperature is theset point, and not the actual reaction temperature, which is a few degrees lower.

The user may enter temperatures other than the recommended setting forqualification or optimization studies.

� Duration of the Deglycosylation Reaction

The digestion procedure has been optimized to deliver deglycosylation of N-Glycansfrom most glycoproteins in 15–60 minutes. The optimal incubation time will varydepending on the specific glycoprotein; those which have proven to be resistant todeglycosylation via conventional enzymatic methods may require longer incubationtimes (up to 60 minutes). For glycoproteins that are comparatively easy todeglycosylate, such as monoclonal antibodies, a 15-minute incubation is generallysufficient.

Often glycoproteins must be denatured to open the protein structure for the enzymeto gain access for cleavage. See the discussion in this section under SampleDenaturation.

It is critical not to exceed a 60-minute incubation, as theCartridge resin bed may dry out, yielding uncertainresults.

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Using the Reagent Volume Calculator Excel spreadsheet file that uses formulas to calculatereagent needs and recipe volumes.

Reagents and other solutions, either provided by the user or supplied with theGlykoPrep-plus Kit, must be prepared prior to dispensing into the Reagent SourcePlate.

NOTE: Some values incorporate a small overagerequired for proper operation of the probes. Forexample, for Required Sample Volume and DenaturationReagent Volume, the calculator automatically adds 5 �l.

Enter the sample information in the green fields at the top of the Sample Info &Reagent Prep tab. Grey fields are calculated values.

Volumes listed in the Reagent Source Plate tab andoptional Labeling Source Plate tab are based on numbersentered into this tab.

� Number of Samples: ________ (actual, green)

Enter the total number of glycoprotein Samples to be processed.

This field is used to calculate the number of columns andvolume of reagents required. Normal processing is donewith complete plate columns, so the number of Samplesshould be 8 to 96 and divisible by 8.

� Number of plate columns used: (calculated value, grey) This is the Number of Samples divided by 8, as only fullcolumns are used. Used to calculate the amount ofreagents to be prepared.

� Target Load (�g): _________ (actual, green)

Enter the desired Glycoprotein Sample Target Load.

The maximum, quantitative Glycoprotein Sample TargetLoad on the RX Cartridge is 50 �g. This value is usedwith the Glycoprotein Sample Concentration to calculatethe total Denatured Sample Load Volume.

� Sample Concentration (mg/ml): _________ (actual, green)

Enter the concentration of the Glycoprotein Samples.

All Glycoprotein Samples will be loaded at the samevolume. To achieve uniform loading across all theCartridges, the Glycoprotein Samples must be adjusted tothe same concentration.

� Required Starting Sample Volume (�l): (calculated value, grey) Calculated from the Sample Target Load and SampleConcentration. Dispense this volume accurately into theGlycoprotein Sample Plate (page 13). This cell will turnred for values greater than 250 �l as this is outside thevolume limit of this parameter.

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� Denaturation Reagent Factor (x:1): _________ (actual, green)

Enter the ratio of Denaturation Reagent to be added to the GlycoproteinSamples; the Denaturation Reagent Factor is the x in the relationship (X:1).

For example, recommended to start: enter 1 for a 1:1mixture to achieve 50% Denaturation Reagent.

� Denaturation Reagent Volume (�l): (calculated value, grey) Calculated from previous entries. This cell will turn redfor values greater than 200 �l, as this is outside thevolume limit of this parameter.

� Denatured Sample Load Volume (�l): (calculated value, grey) Sum of the Glycoprotein Sample Volume and theDenaturation Reagent Volume, less 10 �l to ensureproper filling of the probes.

NOTE: If the sum of the Required Starting SampleVolume plus the Denaturation Reagent Volume less 10 �lis greater than 250 �l, this cell will turn yellow and thevalue will appear as 250 �l, which is the volume limit ofthe syringe.

GlykoPrep-plus Reagent Preparation

Reagents and other solutions, either provided by the user or supplied with the GPPNG-APTS Kit, must be prepared prior to dispensing into the Reagent Source Plate. Pleaserefer to the directions in the GPPNG-APTS Reagent Volume Calculator (Excel files) todetermine the appropriate amount of each to prepare. The “Sample Info and ReagentPrep” tab and the “Reagent Source Plate Setup” tab of the Reagent Volume Calculatormay be printed and taken to a laboratory work station to prepare and then dispensereagents and solutions as shown. The AssayMAP Bravo will dispense the reagents andsolutions from the Reagent Source Plate to begin the sample preparation.

See Preparing for the GlykoPrep-plus Protocols andGlykoPrep-plus Protocols pages 13-31 for preparation ofspecific reagents.

The amount of N-Glycanase (700 �l plus overfill) provided in the kit is sufficient forthe preparation of 96 samples, in a single run or in 4 runs totaling 96 replicates. Ifsmaller runs are desired, it is important to refer to the reagent calculator to confirm theamount of enzyme needed. Additional enzyme is needed to perform greater than 4 runswith a single kit; additional N-Glycanase (WS0278-GP) can be purchased from ProZyme.

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The labware provided in the kit is sufficient for a single run of up to 96samples. If multiple runs are desired, additional labware can be purchased as akit (AM96-NG) or in bulk from ProZyme. Please contact ProZyme for bulkpurchase inquiries.

GPPNG-APTS Kit components are shipped at concentrations that assure maximumstability; some will be diluted prior to dispensing and others will be dispensed directlyfrom the kit containers.

NOTE: Equilibrate all reagents to room temperature, thengently invert to mix.

AssayMAP Bravo Processing Time

The approximate time to process Glycoprotein Samples on the AssayMAP Bravodepends on the specific choices when entering values in the various protocols. Theapproximate times for standard processing are shown in Table 1:

Protocol Fixed Variable Range

(min)The Variable times listed in Table 1 may be influenced bythe following factors:

• Number of columns in use• Digestion incubation time• Denaturation incubation time• Drying time• Load (up to 250 �l at 5 �l/min)• Manual pipetting efficiency• Preheating of the heater before the labeling step

(saves 10 minutes)

1 Plate and Reagent Setup Protocol 15 15 15 - 30

2 RX Cartridge Setup Protocol 1 n/a 1

3 Immobilization and Digestion Protocol 90 30 90 - 120

Drying N-Glycans Prior to Labeling 20 40 20 - 60

4 CU Cartridge Setup Protocol 1 n/a 1

Adding APTS Labeling Reagent n/a 5 - 15 5 - 15

5 Labeling Protocol 70 10 70 - 80

6 Cleanup Protocol 45 n/a 45

Total 247 - 352

(4 ½ to 6 hours)

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Preparing for the GlykoPrep-plus Protocols

Glycoprotein Sample Plate Preparation

Prepare the Glycoprotein Sample Plate with required volumes as described forprocessing on the AssayMAP Bravo.

See: • Sample Info & Reagent Prep tab in the Reagent

Volume Calculator to compute starting volumes. • “GlykoPrep Sample Preparation” above for

information about Glycoprotein Sample concentrationand denaturation conditions.

Dispense the Glycoprotein Samples into either a PCR Plate or a U-Bottom Platedepending on the final Denatured Glycoprotein Sample Volume.

Use the PCR Plate if the Total Volume is 110 μL or less;use the U-Bottom Plate if the Total Volume is110 to 300 μL.

NOTE: Be sure not to introduce bubbles; spin down ifnecessary

NOTE: All Glycoprotein Samples will be loaded at thesame volume. To achieve uniform loading across all theCartridges, the Glycoprotein Samples must be adjusted tothe same concentration.

Reagent Source Plate Preparation

� Print the “Sample Info & Reagent Setup” and “Reagent Source Plate Setup” tabsof the Reagent Volume Calculator.

� Prepare the reagents and solutions needed as described below and in the greysection of the “Sample Info & Reagent Prep” tab.

NOTE: The color cues used here match the protocolcolors in the Agilent application interface.

1. Digestion Buffer

In a separate vial, add the amounts of 25x Digestion Buffer and ultrapure wateras indicated in the Reagent Volume Calculator, and then vortex to mixthoroughly.

� Digestion Buffer may be prepared up to one weekbefore use. Store at 2–8�C.

2. Enzyme Solution

Vortex to mix the vial of N-Glycanase, and then spin down briefly to collect thecontents in the base of the vial.

In a separate vial, add the amounts of Digestion Buffer (prepared above) and

� Enzyme Solution should be prepared on the day ofuse; store at RT.

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N-Glycanase indicated in the Reagent Volume Calculator, and then vortex to mix.

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3. Blocking Reagent (supplied with the Kit)

4. Denaturation Reagent (supplied with the Kit)

5. Finishing Reagent (supplied with the Kit)

� Pipette prepared solutions from the previous step into each well location in theReagent Source Plate as shown in the table in the “Reagent Source Plate Setup”tab.

NOTE: Be sure not to introduce bubbles; spin down ifnecessary

Optional Labeling Source Plate Preparation

No more than one hour prior to use, Labeling Reagent may be prepared inColumn 1 of the Labeling Source Plate and transferred manually by multi-channel pipette to the N-glycan Samples in the Processing Plate.

For preparation of Labeling Reagent, see ManualProcessing - Labeling the N-Glycans with APTS, page 26and Labeling Source Plate tab of the Reagent VolumeCalculator.

Preparation of the AssayMAP Bravo and Associated Equipment

Turn on the AssayMAP Bravo, computer, pump and Inheco controller.

Open the AssayMAP LaunchPad from the desktop.

Under the N-Glycan Sample Prep tab, click on the APTS icon to launch N-GlycanSample Prep: RX digestion & APTS labeling.

Verify that the Wash Station is installed on position#1.

� Connect appropriate tubing from the DI water container to the pump, from thepump to the wash station, and from the wash station to the waste container.

� Fill the wash station source container with DI water (if necessary).� Empty the wash station waste container (if necessary).� Click on “Prime and Wash” to prime the chimneys.� Verify that all chimneys have water flowing.

Verify that the Peltier Thermal Station with PCR Plate Adapter is installed onpostion#4.

Verify that the Orbital Shaking Station is installed on position#9.

Use the LaunchPad web interface to access the ReagentVolume Calculator, GlykoPrep-plus Protocols andsupport documents

NOTE: Ensure that the lines are flushed if changingcomposition of the source.

5 L of purified, deionized water is needed.

During Prime and Wash, syringe tips are washed with 10volumes of DI water, sufficient to eliminate carryoverbetween steps.

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GLYKOPREP-PLUS PROTOCOLS

1. Plate & Reagent Setup Protocol (grey)

2. RX Cartridge Setup Protocol (green)

3. Immobilization and Digestion Protocol (blue)

4. APTS-CU Cartridge Setup Protocol (purple)

5. Labeling Protocol (orange)

6. Cleanup Protocol (red)

In this section, the colored tables give a Deck Layout, aLabware Table and Application Settings for each of theAssayMAP Protocols.

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1 Plate & Reagent Setup ProtocolAssumes full columns are processed

Set the parameters in the Application Settings section:1. Number of Columns to Manage (columns of 8 samples) = _____2. Starting Column of Destination Plate = _____

Third section of the grey graphic above.

Normally Starting Column will be set to 1 unlesspreviously used plates are being reused. This setting willbe the same for all destination plates.

Check the previously prepared Reagent Source Plate for bubbles; centrifuge brieflyto clear bubbles if necessary. Place the Reagent Source Plate on position#5.

From page 13, prepared using the Reagent VolumeCalculator.

Place the empty Seating Station on position#3.

Label a clean PCR Plate as “Processing Plate,” and place it on the Heater StationAdaptor on position#4.

The Processing Plate will contain the released N-glycansafter Immobilization and Digestion.

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Place a Pipette-Tip Box on position#6; REMOVE THE LID. If using less than a full box, the pipette tips must be onthe RIGHT side of the box as you look at the AssayMAPBravo from the front. This is counterintuitive, so pleasedouble check.

This protocol uses 40 pipette tips, which are thendiscarded. Make sure there are at least 40 tips in fullcolumns in the rack of pipette tips.

Label and stack 4 U-Bottom Plates:a. Digestion Buffer (top)b. Blocking Reagentc. Denaturation Reagentd. Finishing Reagent (bottom)

Place the stack on position#7.

Orient the plates so that the A1 wells are pointed towarddeck position#1.

Make sure that the AssayMAP Bravo deck looks like the Deck Layout shown. First section of the grey graphic above; labware is listedin the second section.

Click “Run Protocol 1.” A status line will show when the protocol is complete: "Run complete. Module idle."

Approximate run time is 15 minutes.

WARNING: Do not reach into the AssayMAP Bravo spacewhile it is operating. This will break the security line; theAssayMAP Bravo will make an emergency stop, and maynot be able to be restarted without intervention from aknowledgeable operator.

Proceed to Protocol 2. May delay as much as one hour without affecting resultsbefore proceeding to the next protocol.

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2 RX Cartridge Setup ProtocolAssumes full columns are processed

Rearrange the deck in preparation for processing:1. Remove the Reagent Source Plate from position#2; discard.2. Remove the Seating Station on position#3 and discard the used pipette tips.3. Place the now empty Seating Station on position#2.4. Remove the lid from a Rack of RX Cartridges and place it with its Receiver Plate

on position#6.5. Tape the Cartridge Rack and Receiver Plate to the deck.

Set the parameters in the Application Settings section:

1. Indicate the first column of RX Cartridges to be used in the RX Cartridge Rack inposition#6.

2. Indicate the number of columns to transfer.

Third section of the green graphic above.

NOTE: The first column to be used must be the firstavailable column of Cartridges as the Syringe Headcannot access a buried column of Cartridges. Remainingcolumns are transferred directly after the first column inthe left to right direction.

20

3. Indicate the first column in the Seating Station into which these columns of RXCartridges will be placed.

Normally, the starting column of the RX Cartridge Rackwill match the starting column of the Seating Stationworking across a plate from left to right. For example,columns 1-5 in a run using a full rack would be indicatedby starting column #1 and 5 columns to be moved. Thesecond run using that rack would start with column 6.

Make sure that the AssayMAP Bravo deck looks like the Deck Layout shown. First section of the green graphic above; labware is listedin the second section.

Click on “Run 2 RX Cartridge Setup.” NOTE: The RX Cartridge Setup module can be reversedby clicking on “Undo RX Cartridge Transfer.”

A status line will show when the protocol is complete: “Run complete. Module idle.”

Approximate run time is 1 minute.


Proceed to Protocol 3. May delay as much as one hour before proceeding to thenext protocol without affecting results.

21

3 Immobilization & Digestion Protocol Assumes full columns are processed

Dispense the RX Priming Solution (100 % Acetonitrile) into a 12-Column ReservoirPlate. Fill the channels with the specified volume corresponding to the columns ofRX Cartridges to be processed.

Rearrange the deck in preparation for processing:

1. Remove the RX Cartridge Rack, Receiver Plate and tape from position#6. Replace the lid to protect any remaining RX Cartridges, and store appropriately.

2. Place the 12-Column, Reservoir Plate containing the RX Priming Solution onposition#3, making sure that the filled reservoirs correspond to the RX Cartridgepositions.

Blue section of the printed Reagent Volume Calculator“Sample Info & Reagent Prep” tab.

22

3. Place the Glycoprotein Sample Plate on position#6. The positions of the columns of Glycoprotein Samplesshould correspond to the positions of the columns of RXCartridges.

NOTE: The Glycoprotein Sample Plate can be a PCRPlate or a U-Bottom Plate, depending on the DenaturedSample Load Volume. Use a PCR Plate if this value is110 μL or less; use a U-Bottom Plate if it is between 110and 300 μL.

Set the sample plate type in the “Labware” section, line 6:Select PCR Plate (low volume) or U-Bottom Plate (high volume).

Middle section of the blue graphic above.

WARNING: The correct sample plate type MUST bechosen or the AssayMAP Bravo Head may be damaged.

Set the parameters in the Application Settings section: Third section of the blue graphic above.

1. Denaturant Volume: Enter the volume of Denaturation Reagent to be added toeach Glycoprotein Sample.

Cell D16 in the Reagent Volume Calculator. Themaximum allowable volume is 200 �l.

2. Starting Sample Volume: Enter the starting volume of Glycoprotein Sample to beloaded.

Cell D15 in the Reagent Volume Calculator. Themaximum allowable volume is 250 �l.

3. Denatured Sample Load Volume (calculated value, grey). The Denatured Sample Load Volume is calculatedautomatically (from cells D15 and D16 of the ReagentVolume Calculator, less 10 �l to ensure proper filling ofthe probes) and displayed on the form after the protocolbegins.

The maximum volume that can be loaded on a Cartridgeis 250 �l; if the sum of the Denaturant Volume and theStarting Sample Volume is greater than 260 �l, only 250 �lwill be loaded.

4. Enter the Sample Loading Flow Rate. The recommended flow rate is 5 �l/minute, althoughfaster or slower rates are possible. Flow rates other thanthe recommended rate of 5 �l/minute may affect themaximum quantitative binding capacity.

23

5. Temperature Setpoint for Digestion: Enter the incubation temperature for thedeglycosylation reaction.

Note that this temperature is the set point and not theactual reaction temperature, which is a few degreeslower. The assay has been optimized for a set point of45�C, although laboratory conditions may cause thisoptimum to vary slightly.

The temperature can be monitored on the digital readoutof the STC Controller.

6. Duration of Digestion Step: Enter the duration for the deglycosylation reaction. The level of glycosylation varies from glycoprotein toglycoprotein; see “Duration of the DeglycosylationReaction” page 9.

Make sure that the AssayMAP Bravo deck looks like the Deck Layout. First section of the blue graphic above; labware is listedin the second section.

Click on “Run 3 Immobilization & Digestion.” The protocol will take between 90 and 120 minutesdepending on the sample volume and incubation time.



Proceed to Manual Processing. The Processing Plate (position#4) now contains theN-glycans; remove for drying. The AssayMAP Bravo maybe shutdown at this point.

24

Manual Processing - Drying the N-Glycans prior to labeling

Rearrange the deck in preparation for processing:

� Remove the Processing Plate (PCR plate) from position#4.

� Dry the N-Glycan Samples using a centrifugal evaporator with the heat settingturned to the off position for 20 minutes to 1 hour, or until fully dry. .

The Processing Plate now contains released N-Glycanswith a free reducing end, along with Finishing Reagent.

This is a good time to remove the APTS Solution,Reductant Solution and APTS Catalyst so they can cometo room temperature.

Protocol 4 may be processed simultaneously with Manual Processing.

25

4 CU Cartridge Setup Protocol Assumes full columns are processed

Rearrange the deck in preparation for processing:� Remove all Immobilization and Digestion labware from deck.� Discard used RX Cartridges.

Remove the lid from a Rack of CU Cartridges and place it with its Receiver Plate onposition#6.

May process a partial Rack.

Tape the CU Cartridge Rack and Receiver Plate to the deck.

Place the empty Seating Station in position#2.

26


1. Indicate the first column from which to take the CU Cartridges from Rack inposition#6.

2. Indicate the number of columns to transfer.3. Indicate the first column in the Seating Station where the CU Cartridges are to be

placed.

Third section of the purple graphic above.

NOTE: This must be the first occupied column of the CUCartridge Rack as the Syringe Head cannot access aburied column of Cartridges.

Normally the starting column of the CU Cartridge Rackwill match the starting column of the Seating Stationacross the plate from left to right.

Make sure that the AssayMAP Bravo deck looks like the Deck Layout shown. First section of the purple graphic above; labware is listedin the second section.

Click on “Run 4 CU Cartridge Setup.” NOTE: the CU Cartridge Setup module can be reversedby clicking on “Undo CU Cartridge Transfer”.


Approximate run time is 1 minute.


When the N-glycan Samples are dry, proceed to Manual Processing - Labeling theN-Glycans with APTS.

27

Manual Processing - Labeling the N-Glycans with APTS

Prepare APTS Labeling Reagent.

.

Allow the APTS Solution, Reductant Solution and APTS Catalyst vials to come toroom temperature in the sealed desiccant bag before removing them. Invert each tomix gently. Before opening each vial, flick it or gently tap it on a flat surface todislodge any liquid adhering to the underside of the cap and ensure that thecontents collect at the bottom.

In a separate vial, add the amounts of APTS Solution, Reductant Solution and APTSCatalyst as indicated in the Reagent Volume Calculator, and then vortex to mixthoroughly.

Pipette equal volumes of APTS Labeling Reagent into each well of the first columnof the Labeling Source Plate (PCR plate) as instructed in the Labeling Source Platetab of the Reagent Volume Calculator.

Add the Labeling Reagent:� Add 4.5 μL of Labeling Reagent to the side of each N-Glycan Sample well about

half way down the side of the well.� Tap the plate sharply on the bench and centrifuge briefly if necessary to ensure

that the droplet of Labeling Reagent drops to the bottom of the well.� Cover the plate with the Aluminum Seal provided.

Remove the tabs from the Aluminum Seal, as they can interfere with operation of theAssayMAP Bravo.

Orange section of the printed Reagent Volume Calculator“Sample Info & Reagent Prep” tab.

� Prepare no more than one hour before use

NOTE: APTS Labeling Reagent components arehazardous. Please refer to the Safety Data Sheetsincluded in the Kit or on our website. Perform thisprocedure using appropriate personal safety protection,eyeglasses and nitrile gloves.

NOTE: The APTS Solution is light-sensitive and theReductant Solution and APTS Catalyst are hygroscopic;minimize exposure to air and protect from exposure tolight. The individual reagents may be tightly capped,repackaged with the desiccant in the resealable bag, andfrozen (-20�C) for storage up to 6 months.

Use of the Labeling Source Plate is optional.

Dried N-glycans in Processing Plate.

Visually inspect to make sure there are no bubbles at thebottom of the well.

Supplied with the Kit; film may be cut with scissors ifusing less than the full Kit.

Proceed to Protocol 5.

28

5 Labeling Protocol

Place the sealed Processing Plate containing the N-Glycan Samples plus LabelingReagent on position#4.


� Enter the desired labeling reaction duration.

� Enter the desired labeling reaction temperature.

Third section of the orange graphic above.

Default duration is 60 minutes.

Default temperature is 50�C; allowed temperature range is20–70�C.

The temperature may be monitored on the digital readoutof the STC Controller.

Make sure that the AssayMAP Bravo deck looks like the Deck Layout shown. First section of the orange graphic above; labware islisted in the second section.

29

Click on “Run 5 Labeling.”

The Processing Plate containing N-Glycan Samples is moved from position#4 toposition#9, where it is mixed for one minute. It will remain there until the ThermalStation temperature is within 1�C of the set point (~10 minutes to achieve 50�C). Then the plate will be moved to position#4 and the timer starts. When finished, theplate is moved to position#7 and the protocol ends. The plate movement ensuresthat the N-Glycan Samples are at the desired temperature for the set amount of timewith no additional temperature ramp up or down.

A status line will show when the protocol is complete: “Run complete. Moduleidle.”


Prepare APTS Cleanup Protocol Reagents during the labeling incubation. Red section of the printed Reagent Volume Calculator“Sample Info & Reagent Prep” tab

NOTE: The maximum volume per channel is 7.0 ml.

• APTS Sample Load Buffer:

Add the amount of 5x APTS Sample Load Buffer indicated in the Reagent VolumeCalculator to a glass graduated cylinder using a volumetric pipette. Bring thevolume up to the final volume with HPLC-grade acetonitrile.

Transfer to a similarly sized glass container, cap tightly and swirl gently to mix.

� Prepare on the day of use. Store sealed in a similarlysized, clean, glass container until the Cleanup Protocol inorder to prevent evaporation of the acetonitrile. Precisecontrol of the acetonitrile concentration is important toensure both retention of N-glycans and removal of excessdye during Cleanup.

It is important to prevent preferential evaporation of theacetonitrile, which affects the performance of the HILICseparation.

At the software prompt, prepare for resuspension of the labeling reaction:

1. Move the plate containing the labeled N-Glycan Samples from position#7 to afume hood.

2. Allow the plate to cool for 5 minutes to room temperature.3. Remove the Aluminum Seal.

Remove the Aluminum Seal in a fume hood to avoidinhalation of hazardous reaction products.

NOTE: Be sure to remove all excess aluminum, as it caninterfere with the AssayMAP Bravo grippers and sensors

30

Proceed to Protocol 6.

6 APTS Cleanup Protocol

Remove the CU Cartridge Rack, Receiver Plate and tape from position#6. Replacethe lid to protect any unused Cartridges and store appropriately.

Set the parameters in the Application Settings section: Third section of the red graphic above.

Indicate the Final Eluate Volume. (50 - 100 �l, as needed for the specificanalytical method).

The AssayMAP Bravo elutes the Labeled N-Glycan Samplesquantitatively from the CU Cartridges in 50 �l of water. Any additional elution volume is added in a separate step.

The Labeled N-Glycan Samples are eluted from the CUCartridge as continuous fractions. A 1-minute mixing stepproduces a homogeneous sample, ready for analysis.

31

Place the Processing Plate containing the Labeled N-Glycan Samples on position#4.

Place the previously prepared Cleanup Solutions on the deck, making sure that thefilled reservoirs correspond to the CU Cartridge positions:

1. Place the 12-Column, Reservoir Plate containing CU Washing Solution (6 ml ofAPTS Sample Load Buffer in each reservoir) on position#5.

2. Place the 12-Column, Reservoir Plate containing CU Priming Solution (6 ml ofAPTS Sample Load Buffer in each reservoir) on position#6.

3. Place a 12-Column, Reservoir Plate containing 6 ml of APTS Sample Load Bufferin each reservoir on position#7 .

4. Place a 12-Column, Reservoir Plate containing HPLC-grade water (6 ml in eachreservoir) on position#8.

After removing the Aluminum Seal in a fume hood, eachwell now contains Labeled N-Glycan Samples plusunreacted dye, which will be removed using HILICchromatography.

Place an empty Square, Deep-Well Plate on position#3 for Waste.

Label an empty PCR Plate “CU Eluate” and place it on position#9. At the end of the protocol, the purified labeled N-glycanswill be in the CU Eluate.

Make sure that the AssayMAP Bravo deck looks like the Deck Layout shown. First section of the red graphic above; labware is listed inthe second section.

Click on “Run 6 Cleanup.” The protocol will take approximately 40 minutes.

Status is shown in the status bar.


32

Upon completion of the protocol:

1. Remove the Eluate Collection Plate containing the Labeled N-Glycan Samplesfrom position#9.

2. Proceed directly to analysis, or seal the plate with Aluminum Seal and store at-20�C.

3. Remove all other labware from the deck and discard any remaining solutionsand waste appropriately.


The Labeled N-Glycan Samples are ready for analysis.

NOTE: Waste plate at position#3 contains Acetonitrile;discard according to waste disposal procedures.

Empty the Wash Station waste container.

Qu

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QU

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GU

IDE

Gly

co

pro

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Sa

mp

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late

Pre

pa

ratio

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lyco

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late w

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qu

ired

vo

lum

es u

sing th

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ple

Info

& R

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in th

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lum

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r to co

mp

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starting v

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mes.

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se th

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Reag

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Pre

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the re

agen

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solu

tion

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ed

as describ

ed

belo

w an

d in

the g

rey sectio

n o

f the R

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1.

Dig

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Dig

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rep

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up

to o

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efo

re u

se. Sto

re at 2

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.

In a se

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d th

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� E

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Prin

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ually

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dig

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AP

TS lab

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Verify

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n is in

stalled

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ssary).

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erm

al Station

with

PC

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mb

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f Co

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s) = _

________

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________

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: "Ru

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ceed

to P

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.

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Rem

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n p

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n o

n p

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4.

Rem

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e lid

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of R

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artridges an

d p

lace it w

ith its R

ece

iver P

late o

n p

ositio

n#6.

T

ape th

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X C

artridge R

ack an

d R

ece

iver P

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the d

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.

Set th

e p

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e A

pp

lication

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gs se

ction

:

1.

Ind

icate th

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lum

n o

f RX

Cartrid

ges to

be u

sed

in th

e R

X C

artridge R

ack in

po

sition

#6.

2.

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icate th

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um

ber o

f colu

mn

s to tran

sfer. T

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lum

ns d

irectly

follo

w th

e first co

lum

nin

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d ab

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ft to rig

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irectio

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icate th

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n in

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ating Statio

n in

to w

hich

these

colu

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s of R

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re th

at the A

ssayM

AP

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o d

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loo

ks lik

e th

e D

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n ab

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Click

on

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n 2

RX

Cartrid

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."

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e ap

pro

xim

ate ru

n tim

e is 1

min

ute

. A Statu

s line w

ill sho

w w

hen

the p

roto

col is co

mp

lete

: “Ru

nco

mp

lete

. Mo

du

le id

le.”

Pro

ceed

to P

roto

col 3

.

Qu

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3Im

mo

biliz

atio

n &

Dig

es

tion

Pro

toc

ol

Disp

en

se th

e R

X P

rimin

g So

lutio

n (1

00 %

Ace

ton

itrile) in

to a 1

2-C

olu

mn

Rese

rvo

ir Plate

, corre

spo

nd

ing

to th

e co

lum

ns o

f RX

Cartrid

ges to

be p

roce

ssed

.

Fill th

e ch

ann

els w

ith th

e v

olu

me sp

ecifie

d in

the B

lue se

ction

of th

e p

rinted

Reag

ent V

olu

me C

alculato

r“Sam

ple In

fo &

Reag

ent P

rep

” tab.

Rearran

ge th

e d

eck

in p

rep

aration

for p

roce

ssing:

1.

Rem

ove th

e R

X C

artridge R

ack, R

ece

iver P

late an

d tap

e fro

m p

ositio

n#6. R

ep

lace th

e lid

top

rote

ct any re

main

ing R

X C

artridges an

d sto

re ap

pro

priate

ly.

2.

Place

the 1

2-C

olu

mn

, Rese

rvo

ir Plate

con

tainin

g th

e R

X P

rimin

g So

lutio

n o

n p

ositio

n#3, m

akin

gsu

re th

at the fille

d re

serv

oirs co

rresp

on

d to

the R

X C

artridge p

ositio

ns.

3.

Place

the G

lyco

pro

tein

Samp

le P

late o

n p

ositio

n#6.

Set th

e sam

ple

plate

typ

e in

the “Lab

ware

” sectio

n, lin

e 6

abo

ve:

Sele

ct PC

R P

late (lo

w v

olu

me) o

r U-B

otto

m P

late (h

igh

vo

lum

e).

Qu

ickG

uid

e38

Set th

e p

aramete

rs in th

e A

pp

lication

Settin

gs se

ction

(Th

ird se

ction

of th

e b

lue g

raph

ic abo

ve.):

1.

En

ter th

e D

en

aturan

t Vo

lum

e (ce

ll D16 in

the R

eag

en

t Vo

lum

e C

alculato

r).

2.

En

ter th

e Startin

g Sam

ple

Vo

lum

e (ce

ll D15 in

the R

eag

en

t Vo

lum

e C

alculato

r).

3.

En

ter th

e Sam

ple

Load

ing F

low

Rate

.

4.

En

ter th

e T

em

peratu

re Se

t Po

int fo

r Dig

estio

n.

5.

En

ter th

e D

uratio

n o

f Dig

estio

n ste

p.

Mak

e su

re th

at the A

ssayM

AP

Brav

o d

eck

loo

ks lik

e th

e D

eck

Layo

ut ab

ove.

Click

on

“Ru

n 3

Imm

ob

ilization

& D

igestio

n.”

Th

e p

roto

col w

ill take b

etw

een

90 an

d 1

20 m

inu

tes d

ep

en

din

g o

n th

e sam

ple

vo

lum

e an

d in

cub

ation

time. T

he statu

s line w

ill sho

w w

hen

the p

roto

col is co

mp

lete

: "Ru

n co

mp

lete

. Mo

du

le id

le."

Th

e P

roce

ssing P

late (p

ositio

n#4) n

ow

con

tains th

e N

-gly

cans; re

mo

ve fo

r dry

ing. T

he A

ssayM

AP

Brav

om

ay b

e sh

utd

ow

n at th

is po

int.

Pro

ceed

to M

anu

al Pro

cessin

g.

Ma

nu

al P

roc

es

sin

g - D

ryin

g th

e N

-Gly

ca

ns p

rior to

lab

elin

g

Rearran

ge th

e d

eck

in p

rep

aration

for p

roce

ssing:

•R

em

ove th

e P

roce

ssing P

late (P

CR

plate

) from

po

sition

#4.

•D

ry th

e N

-Gly

can Sam

ple

s usin

g a ce

ntrifu

gal e

vap

orato

r with

the h

eat se

tting tu

rned

off u

ntil fu

llyd

ry (2

0 m

inu

tes to

1 h

ou

r).

Pro

toco

l 4 m

ay b

e p

roce

ssed

simu

ltaneo

usly

with

Man

ual P

roce

ssing.

Qu

ickG

uid

e39

4C

U C

artrid

ge

Se

tup

Pro

toc

ol

Rearran

ge th

e d

eck

in p

rep

aration

for p

roce

ssing:

•R

em

ove all Im

mo

bilizatio

n an

d D

igestio

n lab

ware

from

deck

.•

Discard

use

d R

X C

artridges.

Rem

ove th

e lid

from

a Rack

of C

U C

artridges an

d p

lace it w

ith its R

ece

iver P

late o

n p

ositio

n#6.

Tap

e th

e C

U C

artridge R

ack an

d R

ece

iver P

late to

the d

eck

.

Place

the e

mp

ty Se

ating Statio

n in

po

sition

#2.

Set th

e p

aramete

rs in th

e A

pp

lication

Settin

gs se

ction

:

1. In

dicate

the first co

lum

n fro

m w

hich

to tak

e th

e C

U C

artridges fro

m R

ack in

po

sition

#6.

2. In

dicate

the n

um

ber o

f colu

mn

s to tran

sfer.

3. In

dicate

the first co

lum

n in

the Se

ating Statio

n w

here

the C

U C

artridges are

to b

e p

laced

.

Mak

e su

re th

at the A

ssayM

AP

Brav

o d

eck

loo

ks lik

e th

e D

eck

Layo

ut sh

ow

n ab

ove.

Click

on

"Ru

n 4

CU

Cartrid

ge Se

tup

."

Th

e ap

pro

xim

ate ru

n tim

e is 1

min

ute

. Wh

en

the N

-gly

can Sam

ple

s are d

ry, p

roce

ed

to M

anu

alP

roce

ssing - Lab

elin

g th

e N

-Gly

cans w

ith A

PT

S.

Qu

ickG

uid

e40

Ma

nu

al P

roc

es

sin

g - L

ab

elin

g th

e N

-Gly

ca

ns w

ith A

PT

S

Pre

pare

Labelin

g R

eagen

t (oran

ge se

ction

of th

e p

rinted

Reag

ent V

olu

me C

alculato

r “Samp

le Info

& R

eag

ent

Pre

p” tab

).

� P

rep

are n

o m

ore

than

on

e h

ou

r befo

re u

se

Allo

w th

e A

PT

S Solu

tion

, Red

uctan

t Solu

tion

and

AP

TS C

atalyst v

ials to co

me to

roo

m te

mp

eratu

re in

the se

aled

desiccan

t bag

befo

re re

mo

vin

g th

em

. Invert e

ach to

mix

gen

tly. B

efo

re o

pen

ing e

achvial, flick

it or g

en

tly tap

it on

a flat surface

to d

islod

ge an

y liq

uid

adh

erin

g to

the u

nd

ersid

e o

f the

cap an

d e

nsu

re th

at the co

nte

nts co

llect at th

e b

otto

m.

NO

TE

: AP

TS Lab

elin

g R

eag

en

t com

po

nen

ts are h

azardo

us. P

lease

refe

r to th

e Safe

ty D

ata Sheets o

n o

ur

web

site. P

erfo

rm th

is pro

ced

ure

usin

g ap

pro

priate

perso

nal safe

ty p

rote

ction

, eyeglasse

s and

nitrile

glo

ves.

In a se

parate

vial, ad

d th

e am

ou

nts o

f AP

TS So

lutio

n, R

ed

uctan

t Solu

tion

and

AP

TS C

atalyst as

ind

icated

in th

e R

eag

en

t Vo

lum

e C

alculato

r, and

then

vo

rtex to

mix

tho

rou

gh

ly.

Op

tion

al: Pip

ette

eq

ual v

olu

mes o

f AP

TS Lab

elin

g R

eag

en

t into

each

well o

f the first co

lum

n o

f the

Labelin

g So

urce

Plate

(PC

R p

late) as in

structe

d in

the Lab

elin

g So

urce

Plate

tab o

f the R

eag

en

t Vo

lum

eC

alculato

r.

Ad

d th

e Lab

elin

g R

eag

en

t to d

ried

N-g

lycan

s in th

e P

roce

ssing P

late:

�A

dd

4.5

μL o

f Labelin

g R

eag

en

t to th

e sid

e o

f each

N-G

lycan

Samp

le w

ell ab

ou

t half w

ay d

ow

n th

esid

e o

f the w

ell.

�T

ap th

e p

late sh

arply

on

the b

en

ch to

en

sure

that th

e d

rop

let o

f Labelin

g R

eag

en

t dro

ps to

the

bo

ttom

of th

e w

ell.

�C

over th

e p

late w

ith th

e A

lum

inu

m Se

al pro

vid

ed

. Rem

ove th

e tab

s from

the A

lum

inu

m Se

al, as they can

interfe

re w

ith o

peratio

n o

f the A

ssayM

AP

Brav

o.

Pro

ceed

to P

roto

col 5

.

Qu

ickG

uid

e41

5L

ab

elin

g P

roto

co

l

Place

the se

aled

Pro

cessin

g P

late co

ntain

ing th

e N

-Gly

can Sam

ple

s plu

s Labelin

g R

eag

en

t on

po

sition

#4.

Set th

e p

aramete

rs in th

e A

pp

lication

Settin

gs se

ction

abo

ve:

•E

nte

r the d

esire

d lab

elin

g re

action

du

ration

. Th

e d

efau

lt du

ration

is 60 m

inu

tes.

•E

nte

r the d

esire

d lab

elin

g re

action

tem

peratu

re. T

he d

efau

lt tem

peratu

re is 5

0�C

; allow

ed

tem

peratu

reran

ge is 2

0 - 70�C

.

Mak

e su

re th

at the A

ssayM

AP

Brav

o d

eck

loo

ks lik

e th

e D

eck

Layo

ut sh

ow

n ab

ove.

Click

on

"Ru

n 5

Labelin

g.”

Du

ring th

e lab

elin

g in

cub

ation

, pre

pare

the A

PT

S Cle

anu

p P

roto

col re

agen

ts as ind

icated

in th

e R

ed

sectio

n o

f the p

rinte

d R

eag

en

t Vo

lum

e C

alculato

r “Samp

le In

fo &

Reag

en

t Pre

p” tab

:

•A

PT

S Samp

le Lo

ad B

uffe

r:

� P

rep

are o

n th

e d

ay o

f use

. Store

seale

d in

a similarly

sized

, clean

, glass co

ntain

er u

ntil th

e C

lean

up

pro

toco

l.

Ad

d th

e am

ou

nt o

f 5x A

PT

S Samp

le Lo

ad B

uffe

r ind

icated

in th

e R

eag

en

t Vo

lum

e C

alculato

r to a

glass g

radu

ated

cylin

der u

sing a v

olu

metric p

ipette

.

Brin

g th

e v

olu

me u

p to

the fin

al vo

lum

e w

ith H

PLC

-grad

e ace

ton

itrile.

Tran

sfer to

a similarly

sized

glass co

ntain

er, cap

tigh

tly an

d sw

irl gen

tly to

mix

.

Qu

ickG

uid

e42

At th

e so

ftware

pro

mp

t, pre

pare

for re

susp

en

sion

of th

e lab

elin

g re

action

:

1.

Mo

ve th

e p

late co

ntain

ing th

e lab

ele

d N

-Gly

can Sam

ple

s from

po

sition

#7 to

a fum

e h

oo

d.

2.

Allo

w th

e p

late to

coo

l for 5

min

ute

s to ro

om

tem

peratu

re.

3.

Rem

ove th

e A

lum

inu

m Se

al.

NO

TE

: Rem

ove th

e A

lum

inu

m Se

al in a fu

me h

oo

d to

avo

id in

halatio

n o

f hazard

ou

s reactio

n p

rod

ucts.

Pro

ceed

to P

roto

col 6

.

Qu

ickG

uid

e43

6A

PT

S C

lea

nu

p P

roto

co

l

Rem

ove th

e C

U C

artridge R

ack, R

ece

iver P

late an

d tap

e fro

m p

ositio

n#6. R

ep

lace th

e lid

and

store

app

rop

riately

.

Set th

e p

aramete

rs in th

e A

pp

lication

Settin

gs se

ction

abo

ve:

Ind

icate th

e F

inal E

luate

Vo

lum

e (5

0-1

00 �

l).

Afte

r rem

ovin

g th

e A

lum

inu

m Se

al in a fu

me h

oo

d, p

lace th

e P

roce

ssing P

late co

ntain

ing th

e Lab

ele

dN

-Gly

can Sam

ple

s on

po

sition

#4.

Place

the p

revio

usly

pre

pare

d C

lean

up

Solu

tion

s on

the d

eck

, mak

ing su

re th

at the fille

d re

serv

oirs

corre

spo

nd

to th

e C

U C

artridge p

ositio

ns:

•P

lace th

e 1

2-C

olu

mn

, Rese

rvo

ir Plate

con

tainin

g C

U W

ashin

g So

lutio

n (6

ml e

ach re

serv

oir o

f AP

TS

Samp

le Lo

ad B

uffe

r) on

po

sition

#5.

•P

lace th

e 1

2-C

olu

mn

, Rese

rvo

ir Plate

con

tainin

g C

U P

rimin

g So

lutio

n (6

ml e

ach re

serv

oir o

f AP

TS

Samp

le Lo

ad B

uffe

r) on

po

sition

#6.

•P

lace a 1

2-C

olu

mn

, Rese

rvo

ir Plate

con

tainin

g 6

ml e

ach re

serv

oir o

f AP

TS Sam

ple

Load

Bu

ffer o

np

ositio

n#7.

•P

lace a 1

2-C

olu

mn

, Rese

rvo

ir Plate

con

tainin

g H

PLC

-grad

e w

ater o

n p

ositio

n#8.

Place

an e

mp

ty, Sq

uare

, Deep

-Well P

late o

n p

ositio

n#3 fo

r Waste

.

Place

an e

mp

ty P

CR

Plate

on

po

sition

#9 fo

r Elu

ate.

Qu

ickG

uid

e44

Mak

e su

re th

at the A

ssayM

AP

Brav

o d

eck

loo

ks lik

e th

e D

eck

Layo

ut sh

ow

n ab

ove.

Click

on

"Ru

n 6

Cle

anu

p."

Up

on

com

ple

tion

of th

e p

roto

col:

a.R

em

ove th

e E

luate

Co

llectio

n P

late co

ntain

ing th

e Lab

ele

d N

-Gly

can Sam

ple

s from

po

sition

#9.

Th

e Lab

ele

d N

-Gly

can Sam

ple

s are re

ady fo

r analy

sis.

b.

Pro

ceed

dire

ctly to

analy

sis, or se

al the p

late w

ith A

lum

inu

m Se

al and

store

at -20�C

.

c.R

em

ove all o

ther lab

ware

from

the d

eck

and

discard

any re

main

ing so

lutio

ns an

d w

asteap

pro

priate

ly.

NO

TE

: Waste

plate

at po

sition

#3 co

ntain

s Ace

ton

itrile; d

iscard acco

rdin

g to

waste

disp

osal p

roce

du

res.

Em

pty

the W

ash Statio

n w

aste co

ntain

er.

If the n

ext p

roce

ssing is N

OT

a Gly

ko

Pre

p w

ork

flow

, rem

ove all ap

plian

ces fro

m th

e A

ssayM

AP

Brav

od

eck

.

NO

TE

: Th

e p

rese

nce

of th

e W

ash Statio

n, th

e T

herm

al Station

and

the O

rbital Sh

aker can

cause

dam

age

to th

e A

ssayM

AP

Brav

o H

ead

if the n

ext w

ork

flow

has n

ot b

een

defin

ed

to in

clud

e th

em

.

45

AN

AL

YS

IS O

F L

AB

EL

ED

N-G

LY

CA

NS

Use

stand

ard te

chn

iqu

es, su

ch as C

apillary

Ele

ctrop

ho

resis (C

E), to

analy

ze th

e aq

ueo

us e

luate

con

tainin

g e

lute

d, lab

ele

d N

-gly

cans (se

e T

ips an

d H

ints, b

elo

w).

TIP

S &

HIN

TS

Co

llectio

n P

lates fo

r Dire

ct CE

An

alysis

Assay

MA

P R

acks w

ill ho

ld 2

00 �

l PC

R co

llectio

n tu

bes fo

r use

dire

ctly in

the B

eck

man

Co

ulte

r PA

800

plu

s Ph

armace

utical A

naly

sis Syste

m. T

o co

llect in

PC

R tu

bes, lo

ad o

ne tu

be in

the R

ack co

rresp

on

din

gto

the lo

cation

of e

ach C

artridge in

the e

lutio

n p

late. Stack

the e

lutio

n p

late o

n to

p o

f the R

ack w

ith P

CR

tub

es an

d fo

llow

elu

tion

from

step

4.b

. Fo

r best re

sults, e

lute

samp

les in

100 �

l min

imu

m o

f DI w

ater to

avo

id fu

rther d

ilutio

n p

rior to

CE

analy

sis. En

sure

that th

e e

lute

d sam

ple

s are su

fficien

tly m

ixed

prio

r toC

E.

Reco

very

of th

e D

egly

cosy

lated

Pro

tein

from

the D

igestio

n (R

X) C

artridge

Ofte

n, th

e d

egly

cosy

lated

pro

tein

is analy

zed

to e

valu

ate th

e co

mp

lete

ness o

f degly

cosy

lation

usin

g su

chele

ctrop

ho

retic m

eth

od

s as SDS-P

AG

E o

r micro

fluid

ic lab-o

n-a-ch

ip te

chn

olo

gy. P

lease

con

tact us fo

rgu

idelin

es fo

r elu

ting y

ou

r gly

cop

rote

in fro

m th

e R

X C

artridge.

Calcu

lating th

e M

ass of G

lycan

s Labele

d w

ith A

PT

S

Th

e m

ass of th

e A

PT

S-labele

d N

-gly

can is o

btain

ed

usin

g th

e fo

llow

ing fo

rmu

la:

Gly

canA

PT

S A

PT

S-L

ab

ele

d G

lycan

Mass

+ M

ass=

Mass

Mass A

dd

ed

to G

lycan

Mo

no

isoto

pic

440.9

6468

Averag

e441.5

CE

An

alysis

Th

e A

PT

S label is o

ptim

ized

for an

alysis u

sing C

apillary

Ele

ctrop

ho

resis (C

E). W

e re

com

men

d u

sing

PV

A co

ated

capillarie

s, and

CE

sep

aration

s usin

g an

app

lied

ele

ctric field

of 6

00 V

/cm.

CE

-MS

Mass sp

ectro

metry

and

vario

us ty

pes o

f spectro

scop

ic meth

od

s may

also b

e u

sed

to an

alyze

gly

cans

labele

d w

ith A

PT

S. Th

e lab

el is stab

le u

nd

er acid

ic and

alkalin

e co

nd

ition

s and

do

es n

ot in

terfe

re w

ithth

e actio

n o

f exo

gly

cosid

ases. N

ote

, ho

wever, th

at gly

can stru

cture

s may

no

t be stab

le u

nd

er e

xtre

mes

of p

H. F

or th

is reaso

n, u

sers are

advise

d n

ot to

sub

ject A

PT

S-labele

d g

lycan

s to stro

ngly

acidic o

ralk

aline co

nd

ition

s.

46

Oth

er A

naly

tical Meth

od

s

AP

TS-lab

ele

d g

lycan

s may

also b

e an

alyze

d b

y o

ther sp

ectro

scop

ic meth

od

s (i.e., LC

) usin

g th

ese

wav

ele

ngth

s:

Excitatio

n �

:473 n

mE

missio

n �

:520 n

m

RE

FE

RE

NC

ES

Visit P

roZ

ym

e’s web

site fo

r add

ition

al info

rmatio

n, d

ow

nlo

adab

le p

oste

rs and

instru

ction

al vid

eo

s:

http

://ww

w.p

rozy

me.co

m/g

lyko

prep

Tech

No

te T

NG

P100

Glyk

oP

rep

Gu

ideb

oo

k - G

en

eral tip

s, tricks an

d tro

ub

lesh

oo

ting su

ggestio

ns w

hen

usin

gK

its or m

od

ule

s:

http

://ww

w.p

rozy

me.co

m/d

ocu

men

ts/TN

GP

100

.pd

f

TE

CH

NIC

AL

AS

SIS

TA

NC

E

Pro

Zym

e is co

mm

itted

to d

evelo

pin

g rap

id, au

tom

atable

meth

od

s for g

lycan

analy

sis. Call u

s to d

iscuss p

rod

ucts

in d

evelo

pm

en

t.

If yo

u h

ave an

y q

uestio

ns o

r exp

erie

nce

difficu

lties re

gard

ing an

y asp

ect o

f ou

r pro

du

cts, ple

ase co

ntact u

s:

(800

) 45

7-9

44

4

(US

& C

AN

AD

A)

TO

LL

FR

EE

(510

) 63

8-6

90

0P

HO

NE (5

10

) 63

8-6

91

9F

AX

info

@p

rozym

e.c

om

E-M

AIL w

ww

.pro

zym

e.c

om

WE

B

Pro

Zym

e v

alues cu

stom

er o

pin

ion

s and

con

siders cu

stom

ers an

imp

ortan

t sou

rce fo

r info

rmatio

n re

gard

ing

advan

ced

or sp

ecialize

d u

ses o

f ou

r pro

du

cts. We e

nco

urag

e yo

u to

con

tact us. W

e welco

me y

ou

r suggestio

ns

abo

ut p

rod

uct p

erfo

rman

ce o

r new

app

lication

s and

tech

niq

ues.

47

LIC

EN

SE

TO

US

E

Pu

rchase

of th

is Pro

Zym

e p

rod

uct (th

e "P

rod

uct") fro

m P

roZ

ym

e o

r on

e o

f its auth

orized

distrib

uto

rs com

es w

itha lim

ited

, no

n-tran

sferab

le U

se Lice

nse

to th

e p

urch

aser ("P

urch

aser") u

nd

er P

roZ

ym

e p

aten

ts (the "P

roZ

ym

eP

aten

ts"), wh

ich U

se Lice

nse

is sub

ject to th

e fo

llow

ing te

rms an

d co

nd

ition

s. 1.

Th

e U

se Licen

se is o

nly

for P

urch

aser's in

-ho

use

, in-v

itro u

se o

f the p

urch

ased P

rod

uct. P

urch

aser is no

tlice

nse

d to

use, an

d sh

all no

t use

the P

rod

uct in

any in

vivo

app

lication

and

, for av

oid

ance

of d

ou

bt, is

specifically

pro

hib

ited

from

any u

se o

f the P

rod

uct in

hu

man

s.

2.

Th

e P

rod

uct sh

all no

t be m

ade av

ailable

by P

urch

aser to

any th

ird p

arty in an

y fo

rm, se

parate

ly o

r inco

mb

inatio

ns, w

heth

er fo

r any m

on

etary

or o

ther co

nsid

eratio

n o

r at no

charg

e, with

the fo

llow

ing

exce

ptio

n: th

e P

rod

uct m

ay b

e m

ade av

ailable

to a th

ird p

arty wh

o (a) ag

rees to

be b

ou

nd

by all th

ete

rms an

d re

striction

s of th

is Agree

men

t and

(b) ag

rees to

use

it on

ly fo

r pu

rpo

ses o

f evalu

ation

.

3.

Th

is Use Lice

nse

do

es n

ot in

clud

e th

e rig

ht to

use

the P

rod

uct to

perfo

rm fo

r, or o

ffer co

mm

ercial serv

ices

of an

y k

ind

to, a th

ird p

arty, such

as, with

ou

t limitatio

n, rep

ortin

g th

e resu

lts of an

alyse

s or o

ther activ

ities

to th

e th

ird p

arty for a fe

e o

r oth

er co

mm

ercial con

sideratio

n. P

erso

ns w

ishin

g to

perfo

rm su

ch service

sm

ust o

btain

a sep

arate C

om

mercial Se

rvice

s Licen

se fro

m P

roZ

ym

e, In

c.

4.

Pu

rchase

r is respo

nsib

le fo

r qu

alification

of th

e P

rod

uct fo

r the P

urch

aser's sp

ecific u

se.

Certain

Pro

Zym

e p

rod

ucts in

clud

e co

mp

on

en

ts or re

agen

ts ob

tained

un

der licen

se fro

m o

ther co

mp

anie

s. P

urch

ase of th

ese P

roZ

ym

e p

rod

ucts fro

m P

roZ

ym

e o

r on

e o

f its auth

orize

d d

istribu

tors in

clud

es a lice

nse fro

mth

e o

rigin

ating co

mp

any to

use

the co

mp

on

en

t(s) or re

agen

t(s), bu

t on

ly (1

) with

the o

ther m

aterials in

clud

ed

inth

e P

roZ

ym

e p

rod

uct, an

d (2

) for th

e p

urp

ose

s specified

on

Pro

Zym

e's p

rod

uct, o

n its p

ackag

ing, o

r in th

eacco

mp

anyin

g lite

rature

.

Fo

r a com

ple

te list o

f Pro

Zym

e P

aten

ts, ple

ase v

isit ou

r web

site at h

ttp://w

ww

.pro

zym

e.co

m/p

aten

t.htm

l

Th

e A

ssayMA

P te

chn

olo

gy is lice

nse

d fro

m A

gile

nt T

ech

no

logie

s.

TR

AD

EM

AR

KS

AN

D T

RA

DE

NA

ME

S

Pro

Zym

e, G

lyko

Pre

p, R

apid

-Red

uctiv

e-Am

inatio

n an

d N

-Gly

canase are

tradem

arks o

r registere

d trad

em

arks o

fP

roZ

ym

e, In

c. in th

e U

nite

d States an

d o

ther co

un

tries.

Speed

Vac is a reg

istered trad

em

ark o

f Th

erm

o F

isher Scie

ntific o

r its affiliates.

Milli-Q

is a registe

red

tradem

ark o

f Millip

ore

Co

rpo

ration

in th

e U

nited

States an

d/o

r oth

er co

un

tries.

PR

OD

UC

T U

SE

AN

D W

AR

RA

NT

Y

Term

s and

con

ditio

ns o

f sale m

ay b

e fo

un

d at:

http

://ww

w.p

rozy

me.co

m/te

rms.h

tml

OT

HE

R P

RO

ZY

ME

PR

OD

UC

TS

& K

ITS

A w

ide v

ariety of g

lycoan

alysis pro

du

cts are av

ailable

from

Pro

Zym

e. A

com

plete listin

g is acce

ssible

on

ou

rw

eb

site b

y click

ing o

n G

lyko

Pre

p™

Rap

id Sam

ple

Pre

paratio

n P

latform

:

http

://ww

w.p

rozy

me.co

m

48

OR

DE

RIN

G IN

FO

RM

AT

ION

Fo

r No

rth A

merican

destin

ation

s: telep

ho

ne o

rders m

ay b

e p

laced

betw

een

8:00

am an

d 5

:00 p

m P

acific Tim

e. T

ele

fax o

r e-mail o

rders m

ay b

e se

nt o

r messag

es reco

rded

anytim

e.

(800) 4

57-9

444 (U

S &

CA

NA

DA

)T

OLL

FR

EE

(510) 6

38-6

900

PH

ON

E (510) 6

38-6

919

FA

X

info

@p

rozy

me.co

mE-M

AIL w

ww

.pro

zym

e.co

mW

EB

Ou

tside N

orth

Am

erica:

A list o

f Pro

Zym

e’s d

istribu

tors, w

ith co

ntact in

form

ation

, may

be fo

un

d at:

http

://ww

w.p

rozy

me.co

m/d

istribu

tors.h

tml

If there is n

o d

istribu

tor in

yo

ur area, in

structio

ns fo

r placin

g an

inte

rnatio

nal o

rder m

ay b

e fo

un

d at:

http

://ww

w.p

rozy

me.co

m/o

rderin

g.h

tml

49

38

32

Bay

Ce

nte

r Pla

ce

Hay

ward

, CA

94

54

5-3

61

9 (8

00

) 45

7-9

44

4T

OL

L F

RE

E

info

@p

rozym

e.c

om

E-MA

IL (51

0) 6

38

-69

00

PH

ON

E

W

EB

ww

w.p

rozym

e.c

om

(51

0) 6

38

-69

19

FA

X

Rev. A

B