Applied Biosystems iTRAQ Reagents

Applied Biosystems iTRAQTM Reagents

Amine-Modifying Labeling Reagents for Multiplexed Relative and Absolute Protein Quantification

Chemistry Reference Guide

Not for Use in Diagnostics

Copyright 2004, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. This product and its use are covered by one or more pending United States and by foreign patent applications. TRADEMARKS: Applied Biosystems, Analyst, MicroIonSpray, POROS, QSTAR, and TurboIonSpray are registered trademarks, and AB (Design), Applera, BioAnalyst, Explorer, IonSpray, iTRAQ, NanoSpray, and TURBO V are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. Q TRAP is a registered trademark of Applied Biosystems/MDS Sciex, which is a joint venture between Applera Corporation and MDS, Inc. Teflon is a registered trademark of E.I. Du Pont de Nemours and Company. Probot, Ultimate, and Switchos are trademarks of LC Packings ? a Dionex Company. Tween 20 is a registered trademark of ICI Americas, Inc. Milli-Q and ZipTip are registered trademarks of Millipore Corporation. PicoTips is a registered trademark of New Objectives, Inc. NP is a registered trademark of Shell Chemical Company. Triton is a registered trademark of Union Carbide Corporation. PEEK is a trademark of the Victrex Corporation. All other trademarks are the sole property of their respective owners.

Part Number 4351918 Rev. A 05/2004

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Contents

Preface

How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

Safety Information

Chapter 1

Introduction to iTRAQTM Reagents Chemistry

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 iTRAQTM Reagent Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4 User-Supplied and Kit Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6

Chapter 2

iTRAQTM Reagents Protocol

Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 Running the Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6 Preparing the Sample Mixture for LC/MS/MS Analysis . . . . . . . . . . . . 2-9

Chapter 3

Analyzing with a QSTAR? System

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2 Setting Up LC Conditions for the QSTAR? System . . . . . . . . . . . . . . . 3-4 Setting Up Software the First Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Setting Software Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8

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Chapter 4

Analyzing with a Q TRAP? System

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2 Setting Up LC Conditions for the Q TRAP? System . . . . . . . . . . . . . . 4-4 Setting Up Software the First Time . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6 Setting Software Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7

Chapter 5

Analyzing with a 4700 Analyzer

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2 Setting Acquisition Method Parameters . . . . . . . . . . . . . . . . . . . . . . . 5-6 Manually Calculating Relative Quantitation . . . . . . . . . . . . . . . . . . . . 5-11

Appendix A Modifying the iTRAQTM Reagents Protocol

Protocol Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2 Guidelines for Modifying the Protocol . . . . . . . . . . . . . . . . . . . . . . . . . A-2 Testing the Modified Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-7

Appendix B Six-Protein Mix Specifications

Appendix C Preparing the Sample Mixture for Analysis

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-2 Preparing a Simple Sample Mixture for LC/MS/MS Analysis Using Cation Exchange Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-4 Preparing a Complex Sample Mixture for LC/MS/MS Analysis Using HighResolution Cation Exchange Chromatography . . . . . . . . . . . . . . . . . . C-8

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Preface

How to Use This Guide

Purpose of This Guide

This reference guide provides supplementary reference information about labeling peptides with iTRAQTM Reagents and then performing an iTRAQTM Reagents analysis. It:

? Explains iTRAQTM Reagents chemistry ? Describes the iTRAQTM Reagents kits and kit materials ? Describes how to test, run, and modify the iTRAQTM Reagents

protocol

? Provides sample handling guidelines

? Provides suggested LC/MS/MS conditions

? Summarizes the software tools available for performing quantification

Audience This guide is intended for personnel using iTRAQTM Reagents

labeling to quantity a protein or peptide in up to four different samples in one LC/MS/MS analysis.

Assumptions

This guide assumes that you have a working knowledge of an Applied Biosystems/MDS SCIEX QSTAR? Hybrid LC/MS/MS

Quadrupole TOF system, an Applied Biosystems/MDS SCIEX Q TRAP? System, or an Applied Biosystems 4700 Proteomics

Discovery System, if applicable.

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