Waters GlycoWorks High-throughput Sample Preparation Kit User Manual

[ CARE AND USE MANUAL ]

GlycoWorks High-throughput Sample Preparation Kit

CONTENTS

I. INTRODUCTION

II. STORAGE AND DISPOSAL OF PLATES

III. ABBREVIATIONS

IV. USING THE GLYCOWORKS HIGH-THROUGHPUT KIT

V. PREPARING LABELED GLYCANS FOR HILIC-FLR WITH

AN ACQUITY UPLC BEH GLYCAN COLUMN

VI. REFERENCES

VII. ORDERING INFORMATION

I. INTRODUCTION

This document provides information regarding the general care and

use of the Waters GlycoWorks™ High-throughput Sample Preparation

Kit for release of N-glycans. T his protocol can be used as a guideline

and is validated using IgG but can also be used and optimized for

other N-linked glycoproteins. This sample preparation offers an

inclusive protocol that allows the user to source many of the critical

components needed to perform the preparation from one vendor.

General Guideline of Sample Preparation from Glycoprotein to
enrich FLR Labeled Glycans Using Reductive Amination Reaction.

Glycoproteins

Denaturation and Deglycosylation

1

2

3

4

RapiGest™ (if necessary)
DTT Reduction (if necessary),
IAM Alkylation (if necessary)
Deglycosylation by PNGase F

Extraction of Released Glycans

GlycoWorks HILIC µElution Plate

FLR Labeling Reaction of Glycans

Excess Labeling Reagent Removal

GlycoWorks HILIC µElution Plate

FLR Labeled Glycans

MALDI MS

LC-FLR

LC-FLR-MS

The 96-well plate is good for 48 analyses.

For expected results using the protocol with the control standard,

please see Reference 1.

The guide provides the following:

■

An instruction template for new users that highlights the use of a

control standard (included in the kit) for first time validation as

well as subsequent troubleshooting

■

Protocol for LC/FLR and LC/FLR/MS

■

Tips and tricks throughout each section to help aid in the success

■

An online training video is also available (see Reference 3)

GlycoWorks High-throughput Sample Preparation Kit

If using MALDI please refer to Product Solution, Literature Code 720004660EN

Figure 1: Sample Preparation from Glycoprotein to Labeled Glycans

1

[ CARE AND USE MANUAL ]

Table 1: Kit Components

Kit Component Purpose

GlycoWorks HILIC
µElution Plate

GlycoWorks Glycoprotein
Control Standard

RapiGest SF An enzyme friendly detergent used

Manifold Waste Tray For collection of non-releasing glycans

GlycoWorks Reagent Kit Contains ready to use reagents for both

Extract and clean up of free or FLR
labeled glycans

■

Capture of glycans

■

Removing contaminants like salt and

detergents from hydrophilic analytes,
such as carbohydrates, prior to MS

■

Removing excess

labeling reagent

■

The 5 mg sorbent was test to have

maximum binding capacity for 200 µg
glycans

Unlabeled IgG (100 µg/lyophilized) to
use as a control for process validation and
troubleshooting

to denature the glycoproteins prior to
deglycosylation reaction. For stability
purposes, there is enough RapiGest included
for initial use of the kit with 5 samples, more
may need to be ordered for processing all 46
samples.

steps. This is disposable but can be
cleaned and used several times before
ac quiring a new one as long as it is rinsed
out.

denaturation, reduction, and alkylation as
well as raw materials for FLR tagging.

II. STORAGE AND DISPOSAL OF PLATES

Plates stored in their original sealed pouch remain stable for long

periods. To store unused plates in open pouches, squeeze the air out

of the pouch, fold over the open end of the pouch twice, seal with

tape, and store in a desiccator.

Note: Dispose of used plates safely in accordance with applicable

government or local regulations

III. ABBREVIATIONS

IV. USING THE GLYCOWORKS HIGH-THROUGHPUT KIT

Below, are general guidelines for utilizing the GlycoWorks High-

throughput Kit for complete extraction, purification and concentration

of N-linked glycans validated using IgG. Also included is a check list of

accessory materials that will be needed to complete the protocol from

start to finish as well as tips and tricks throughout the instructions.

Table 2: Checklist of Additional Materials Needed Before You Begin

Material
Description

Ammonium
bicarbonate

Ammonium acetate

Formic acid (to make
1% [v/v] formic acid,
prepared in 50%
acetonitrile)

15,000 units
(500,000 units/mL)
PNGase F*

Acetonitrile

25 mM ammonium
bicarbonate in water,
pH 8–9

100 mM ammonium
acetate in 5% [v/v]
acetonitrile, pH 7

85% [v/v] acetonitrile See Above Yes No

96-well collection plate

2-AB** Sigma P/N A89804 Yes No

µElution plate
manifold

SPE vacuum pump

Positive pressure
manifold

Centrifugal vacuum
evaporator

Heat block N/A Ye s No

Recommended

Suppliers

Sigma Aldrich

Cat No. O9830

Sigma Aldrich

Cat No. 73594 FLUKA

Sigma Aldrich

Cat No. F0507

New England Biolabs

Cat. No. P0704S

Sigma Aldrich

Cat No. 360457

See Above Yes No

See Above Yes No

Waters P/N

186 002481

Waters P/N

186 001831

115, 60 Hz:

Waters P/N 176002986

Waters P/N

186 006961

N/A Ye s No

Check List:

Circle

Yes No

Yes No

Yes No

Yes No

Yes No

Yes No

Yes No

Yes No

Yes No

DMSO Dimethylsulfoxide IAM Iodoacetamide

DTT Dithiothreitol 2AB 2-aminobenzamide

GlycoWorks High-throughput Sample Preparation Kit 2

* PNGase F comparison table: https://www.neb.com/tools-and-resources/

usage-guidelines/glycobiology-unit-conversion-chart

**Inclusive Kits are also offered by ProZyme, Ludger, and Sigma covered

under US Patent No. 5,747,347 and its foreign equivalents.