1、iTRAQ Reagents 8plex Amine-Modifying Labeling Reagents for Multiplexed Relative and Absolute Protein Quantitation Protocol For Research Use Only Not for Use in Diagnostics 2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is be
2、ing used under license. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. AB Sciex Pte. Ltd. assumes no responsibility for any errors that may appear in this document. AB SCIEX PTE. LTD. DISCLAIMS ALL W
3、ARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL AB SCIEX PTE. LTD. BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY , OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCI
4、DENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. NOTICE TO PURCHASER: LIMITED LICENSE The purchase of this product includes a limited, non-transferable license to use this amount of the produ
5、ct to practice the labeling methods using encoded isobaric and/or isomeric tagging reagents which may be claimed in pending and planned U.S. and international patent applications exclusively licensed to AB Sciex Pte. Ltd, and patents issuing therefrom, solely for the purchasers research activities.
6、No use in diagnostic procedures is authorized. The license granted herein is personal to the original purchaser and may not be transferred to any other party outside the purchasers company. No other right or license is conveyed or granted either expressly, by implication or by estoppel, to resell or
7、 repackage this product. Certain other restrictions and limitations may apply as set forth in warranty statements and other documentation provided with the purchase of iTRAQ reagent technology.Further information can be obtained by contacting the Director of Licensing, c/o AB Sciex Pte. Ltd., 110 Ma
8、rsh Rd., Foster City, California 94404, USA. Part Number 4375249 Rev. C 08/2010Contents iTRAQ Reagents 8plex Protocol iii Preface Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v How to Obtain More Information . . . . . . . . . . . . .
9、. . . . . . . . . . . . . . . . . . x How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x Chapter 1 Introduction to iTRAQ Reagents 8plex Chemistry Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10、. 1-2 Assay, iTRAQ Reagent 8plex, and Buffer Kits . . . . . . . . . . . . . . . . . 1-4 Kit Materials Packaged with the One Assay Kit . . . . . . . . . . . . . . . . . . 1-5 Kit Materials Packaged with the iTRAQ Reagent -8plex Multi-Plex, 25 U, and 50 U Kits . . . . . . . . . . . . . . . . . . . . .
11、 . . . . . . . . . . . . . . . . . . . 1-7 Kit Materials Packaged with the Buffer Kit . . . . . . . . . . . . . . . . . . . . . . 1-9 User-Supplied Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12、. . . . . . . . . . . . . . . . . . 1-12 Chapter 2 iTRAQ Reagents 8plex - Labeling Protocol Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 Testing the Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4 Ru
13、nning the Labeling Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7 Reducing the Proteins and Blocking Cysteine . . . . . . . . . . . . . . . . 2-7 Digesting the Proteins with Trypsin . . . . . . . . . . . . . . . . . . . . . . . . 2-8 Labeling the Protein Digests with the
14、iTRAQ Reagents 8plex . . 2-9 Combining the iTRAQ Reagents 8plex-Labeled Samples . . . . 2-10iv iTRAQ Reagents 8plex Protocol Chapter 3 iTRAQ Reagents 8plex - Sample Mixture Analysis Preparing the Sample Mixture for LC/MS/MS Analysis . . . . . . . . . . . . 3-2 Performing Cation-Exchange Chromatograp
15、hy . . . . . . . . . . . . . . 3-3 Guidelines and Tutorials for LC/MS/MS Analysis . . . . . . . . . . . . . . . . . 3-4v iTRAQ Reagents 8plex Protocol Preface This preface covers: Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v How to Obtain M
16、ore Information . . . . . . . . . . . . . . . . . . . . . . . . . . x How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Safety Safety Alert Words Four safety alert words appear in our user documentation at points in the document where you need to be aware of
17、relevant hazards. Each alert wordIMPORTANT, CAUTION, WARNING, DANGERimplies a particular level of observation or action, as defined below. Definitions IMPORTANT! Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. Indicates
18、a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. Indicates an imminently hazardous situatio
19、n that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations.vi Preface iTRAQ Reagents 8plex Protocol Chemical Hazard Warning CHEMICAL HAZARD. Some of the chemicals used with our instruments and protocols are potentially hazardous a
20、nd can cause injury, illness, or death. Chemical Safety Guidelines To minimize the hazards of chemicals: Read and understand the Material Safety Data Sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. (See “About MSDSs” o
21、n page vi.) Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. Minimize the inhalation of chemicals. Do not leave chemical container
22、s open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the MSDS. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturers cleanup procedures as recommended in the MSDS. Comply with all local, state/provi
23、ncial, or national laws and regulations related to chemical storage, handling, and disposal. About MSDSs Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with shipments of hazardous chemicals to new customers. They also provide MSDSs with the first shipment of a hazardous ch
24、emical to a customer after an MSDS has been updated. MSDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely. Each time you receive a new MSDS packaged with a hazardous chemical, be sure to replace the appropriate MSDS in your files.vii Safety i
25、TRAQ Reagents 8plex Protocol Obtaining MSDSs You can obtain the MSDS for any chemical supplied with this kit at Note: For the MSDSs of chemicals not distributed with this kit, contact the chemical manufacturer. Chemical Waste Hazards HAZARDOUS WASTE. Refer to Material Safety Data Sheets and local r
26、egulations for handling and disposal. CHEMICAL WASTE HAZARD. Wastes produced by our instruments are potentially hazardous and can cause injury, illness, or death. CHEMICAL STORAGE HAZARD. Never collect or store waste in a glass container because of the risk of breaking or shattering. Reagent and was
27、te bottles can crack and leak. Each waste bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles.viii Preface iTRAQ Reagents
28、 8plex Protocol Chemical Waste Safety Guidelines To minimize the hazards of chemical waste: Read and understand the Material Safety Data Sheets (MSDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste. Provide primary and se
29、condary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) Minimize conta
30、ct with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adeq
31、uate ventilation (for example, fume hood).For additional safety guidelines, consult the MSDS. Handle chemical wastes in a fume hood. After emptying the waste container, seal it with the cap provided. Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practi
32、ces and local, state/provincial, or national environmental and health regulations. Waste Disposal If potentially hazardous waste is generated when you operate the instrument, you must: Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrate
33、s used in your laboratory. Ensure the health and safety of all personnel in your laboratory. Ensure that the instrument waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. IMPORTANT! Radioactive or biohazardous materials m
34、ay require special handling, and disposal limitations may apply.ix Safety iTRAQ Reagents 8plex Protocol Biological Hazard Safety BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Fo
35、llow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate sa
36、fety equipment (for example, physical containment devices). Individuals should be trained according to applicable regulatory and company/institution requirements before working with potentially infectious materials. Read and follow the applicable guidelines and/or regulatory requirements in the foll
37、owing: U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories (stock no. 017-040-00547-4; http:/bmbl.od.nih.gov) Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR1910.1030; http:/www.access.gpo.gov/ nara/cfr/
38、waisidx_01/ 29cfr1910a_01.html). Your companys/institutions Biosafety Program protocols for working with/handling potentially infectious materials. Additional information about biohazard guidelines is available at: http:/www.cdc.govx Preface iTRAQ Reagents 8plex Protocol How to Obtain More Informati
39、on Related Documentation T h e xTRAQ Family of Amine -Modifying Labeling Reagents for Multiplexed Relative and Absolute Quantification: Chemistry Reference Guide. iTRAQ Reagents8plex Chemistry Quick Reference Card (PN 4383502) Technical and Application Notes For the portable document format (PDF) ve
40、rsions of the chemistry reference guide, this protocol, and the quick reference card, go to http:/, then click the link for Support. Click the literature link and perform a literature search. For technical and application notes, see “How to Obtain Support” on page x. Obtaining Information from the S
41、oftware Help System The ProteinPilot Software has a Help system that describes how to use each feature of the user interface. Access the Help system by doing one of the following: Click in the toolbar of the software window Select the Help tab Press F1 How to Obtain Support We are committed to meeti
42、ng the needs of your research. Please go to and go to the Support tab for local support information. Contacting Te c h n i c a l Support in North America To contact technical support: By telephone: Dial 1.877.740.2129 By fax: Dial 1.650.627.28031-1 iTRAQ Reagents 8plex Protocol 1 Introduction to iT
43、RAQ Reagents 8plex Chemistry 1 This chapter covers: Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Assay, iTRAQ Reagent 8plex, and Buffer Kits . . . . . . . . . . . 1-4 Kit Materials Packaged with the One Assay Kit . . . . . . . . . . . . . 1-5 Kit
44、 Materials Packaged with the iTRAQ Reagent 8plex Multi- Plex, 25 U, and 50 U Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7 Kit Materials Packaged with the Buffer Kit . . . . . . . . . . . . . . . . . 1-9 User-Supplied Materials . . . . . . . . . . . . . . . . . . . . . . . .
45、 . . . . . . . 1-10 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-12iTRAQ Reagents 8plex Protocol 1-2 Overview Overview iTRAQ Reagents 8plex are provided as a set of eight, isobaric reagents: iTRAQ Reagents 8plex - 113, 114, 115, 116, 117, 118, 119, 1
46、21 . The use of eight reagents allows multiplexing of up to eight different samples in a single LC/MS/MS experiment. Key Features Using iTRAQ Reagents 8plex to label peptides allows you to: Analyze normal, diseased, and drug-treated states in the same experiment or time-course study. Run duplicate,
47、triplicate, and quadruplet analyses of the same sample in one experiment. Label multiple peptides in a peptide digest (including those from proteins with post-translational modifications) in 2 hours at room temperature. Label multiple peptides per protein, increasing confidence in identification and
48、 quantitation. For easy data interpretation for relative and absolute protein quantitation, analyze samples using ProteinPilot Software and one of the following AB SCIEX instruments: 5600 Triple TOF Systems Q T R A P Systems 5800 MALDI TOF/TOF Instruments There is no iTRAQ Reagent - 120. The phenyla
49、lanine immonium ion mass is 120.iTRAQ Reagents 8plex Protocol 1-3 Overview For More Information iTRAQ Reagent 8plex Chemistry Reference Guide Refer to the xTRAQ Family of Amine -Modifying Labeling Reagents for Multiplexed Relative and Absolute Quantification: Chemistry Reference Guide for supplementary information on iTRAQ Reagent chemistry. Software Help Syst
