Waters GlycoWorks Single Use Sample Preparation Kit User Manual

[ CARE AND USE MANUAL ]

GlycoWorks Single Use Sample Preparation Kit

CONTENTS

I. INTRODUCTION

II. STORAGE AND DISPOSAL OF CARTRIDGES

III. ABBREVIATIONS

IV. USING THE GLYCOWORKS SINGLE USE 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™ Single Use 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.

The Single Use Catridge Pack contains 20 1cc catridges, good for

10 analyses.

For comparison results using this protocol versus the 96 well plate

and for recovery information with the control standard

1,2

.

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

Glycoproteins

Denaturation and Deglycosylation

1

2

3

4

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

RapiGest™ (if necessary)

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

Extraction of Released Glycans

GlycoWorks HILIC 1 cc Cartridge

FLR Labeling Reaction of Glycans

Excess Labeling Reagent Removal

GlycoWorks HILIC 1 cc Cartridge

FLR Labeled Glycans

MALDI MS

LC-FLR

LC-FLR-MS

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

GlycoWorks High-throughput Sample Preparation Kit

3

Figure 1: Sample Preparation from Glycoprotein to Labeled Glycans

1

[ CARE AND USE MANUAL ]

Table 1: Kit Components

Kit Component Purpose

GlycoWorks
HILIC 1 cc
Cartridge

GlycoWorks
Glycoprotein
Control
Standard

RapiGest SF An enzyme friendly detergent used to denature

Manifold
Waste Tray

GlycoWorks
Reagent Kit

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 10 mg sorbent was test to have maximum

binding capacity for 400 µg glycans

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

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 10 samples.

For collection of non-releasing glycans steps.
This is disposable but can be cleaned and used
sever al times befo re acquiring a new one as long
as it is rinsed out.

Contains ready to use reagents for both
denaturation, reduction, and alkylation as well as
raw materials for FLR tagging.

II. STORAGE AND DISPOSAL OF CARTRIDGES

Cartridges stored in their original sealed pouch remain stable for

long periods. For long term storage of unused cartridges, keep

cartridges sealed and in a desiccator.

Note: Dispose of used cartridges safely in accordance with applicable

government or local regulations

III. ABBREVIATIONS

DMSO Dimethylsulfoxide IAM Iodoacetamide

DTT Dithiothreitol 2AB 2-aminobenzamide

IV. USING THE GLYCOWORKS SINGLE USE KIT

Below, are general guidelines for utilizing the GlycoWorks Single

Use 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

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

SPE vacuum pump

Centrifugal vacuum
evaporator

Heat block N/A Yes No

Fisherbrand Premium

1.5 mL MCT graduated,
RNase/DNase free

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

usage-guidelines/glycobiology-unit-conversion-chart

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

115, 60 Hz:

Waters P/N 176002986

N/A Yes No

Fisher Scientific Cat No.

05- 4 08 -129

Check List:

Circle

Yes No

Yes No

Yes No

Yes No

Yes No

Yes No

Yes No

GlycoWorks Single Use Sample Preparation Kit 2

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

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