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iTRAQ+Protocol-ABI


Applied Biosystems iTRAQ? Reagents
Amine-Modifying Labeling Reagents for Multiplexed Relative and Absolute Protein Quantitation

Protocol
For Research Use Only Not for Use in Diagnostics

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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 is a registered trademark, and AB (Design), Applera, and iTRAQ are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. QSTAR and Q TRAP are a registered trademarks of Applied Biosystems/MDS Sciex, which is a joint venture between Applera Corporation and MDS, Inc. Tween is a registered trademark of ICI Americas, Inc. Milli-Q and ZipTip are registered trademarks of Millipore Corporation. NP is a registered trademark of Shell Chemical Company. Triton is a registered trademark of Union Carbide Corporation. All other trademarks are the sole property of their respective owners.

Part Number 4350831 Rev. C 10/2004

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Contents
Safety Information Chapter 1 Introduction to iTRAQ? Reagents Chemistry
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Chapter 2

iTRAQ? Reagents Protocol
Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 Running the Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5 Preparing the Sample Mixture for LC/MS/MS Analysis . . . . . . . . . . . . 2-9

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Safety Information
Safety Alert Words
Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards. Each alert word–IMPORTANT, CAUTION, WARNING, DANGER–implies a particular level of observation or action, as defined below:
IMPORTANT! – Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical.

– Indicates 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 situation that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations.

Chemical Hazard Warning

CHEMICAL HAZARD. Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury, illness, or death.

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Safety Information

Chemical Safety Guidelines

To minimize the hazards of chemicals: ? Read and understand the Material Safety Data Sheets (MSDS) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. (See “About MSDSs” on 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 containers 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 manufacturer’s cleanup procedures as recommended on the MSDS. ? Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. 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 chemical 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.

About MSDSs

Obtaining MSDSs

You can obtain from Applied Biosystems the MSDS for any chemical supplied by Applied Biosystems. This service is free and available 24 hours a day. To obtain MSDSs: 1. Go to https://docs.appliedbiosystems.com/msdssearch.html 2. In the Search field, type in the chemical name, part number, or other information that appears in the MSDS of interest. Select the language of your choice, then click Search.

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3. Find the document of interest, right-click the document title, then select any of the following: ? Open – To view the document ? Print Target – To print the document ? Save Target As – To download a PDF version of the document to a destination that you choose 4. To have a copy of a document sent by fax or e-mail, select Fax or Email to the left of the document title in the Search Results page, then click RETRIEVE DOCUMENTS at the end of the document list. 5. After you enter the required information, click View/Deliver Selected Documents Now.

Chemical Waste Hazard Chemical Waste Safety Guidelines

CHEMICAL WASTE HAZARD. Some wastes produced by the operation of the instrument or system are potentially hazardous and can cause injury, illness, or death. 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 secondary 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 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 containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the MSDS. ? Handle chemical wastes in a fume hood.

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Safety Information

? 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 practices 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 substrates 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 may require special handling, and disposal limitations may apply.

Biological Hazard Safety

BIOHAZARD. Biological samples such as tissues, body fluids, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective eyewear, clothing, and gloves. Read and follow the guidelines in these publications: ? 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 CFR§1910.1030; http://www.access.gpo.gov/nara/cfr/ waisidx_01/29cfr1910a_01.html). Additional information about biohazard guidelines is available at: http://www.cdc.gov

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Introduction to iTRAQ? Reagents Chemistry 1
This chapter covers:

1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

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Chapter 1 Introduction to iTRAQ? Reagents Chemistry

Overview
Applied Biosystems iTRAQ? Reagents are provided as a set of four, isobaric (same mass) reagents: ? ? ? ? iTRAQ? Reagent 114 iTRAQ? Reagent 115 iTRAQ? Reagent 116 iTRAQ? Reagent 117

The use of four reagents allows multiplexing of up to four different samples in a single LC/MS/MS experiment.

Key Features

Using iTRAQ? Reagents to label peptides allows you to: ? Analyze normal, diseased, and drug-treated states in the same experiment or time-course study ? Run duplicate or triplicate analyses of the same sample in one experiment ? Label multiple peptides in a peptide digest, including those from proteins with post-translational modifications, in one hour at room temperature. ? Label multiple peptides per protein, increasing confidence in identification and quantitation The Pro QUANT Software for the Applied Biosystems/MDS SCIEX QSTAR? Hybrid LC/MS/MS Quadrupole TOF system and the Q TRAP? system provides easy data interpretation for relative and absolute quantitation.

Available iTRAQ Reagents Kits
?

? iTRAQ? Reagents Methods Development Kit – Contains iTRAQ? Reagents 114 and 117 and sufficient material to develop a robust protocol and verify that your sample preparation protocol does not interfere with labeling and digestion. First orders also contain cation-exchange buffers, cartridge, and hardware. ? iTRAQ? Reagents Multiplex Kit – Contains sufficient iTRAQ? Reagents (114, 115, 116, and 117) for 10 duplex, 6 three-plex, or 5 four-plex experiments.

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Workflow

Workflow
In the iTRAQ? Reagents labeling protocol, you reduce, cysteine block, digest, and label each sample in a single tube. The single-tube process eliminates potential sample loss in individual samples that may cause inaccuracies in quantitation. Then you combine all iTRAQ? Reagent-labeled samples into one sample mixture for LC/MS/MS analysis. If losses occur during analysis, each sample experiences the same loss and the ratios are preserved. Figure 1-1 summarizes the iTRAQ? Reagents workflow for a duplex-type experiment. Up to four samples can be prepared and analyzed in a single experiment.
Control sample Test sample

Reduce, cysteine block, and digest

Reduce, cysteine block, and digest

Label with an iTRAQTM Reagent

Label with a different iTRAQTM Reagent

Combine the iTRAQ Reagent-labeled control and test sample digests into one sample mixture

Clean up or (optionally) perform highresolution fractionation

Analyze the mixture by LC/MS/MS for protein identification and quantitation

Figure 1-1

Overview of iTRAQ? Reagents methodology

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Chapter 1 Introduction to iTRAQ? Reagents Chemistry

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iTRAQ? Reagents Protocol

iTRAQ? Reagents Protocol
This chapter covers:

2

2

Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 Running the Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5 Preparing the Sample Mixture for LC/MS/MS Analysis . . . . . . . 2-9

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Chapter 2 iTRAQ? Reagents Protocol

This chapter describes preparing your sample, testing the iTRAQ? Reagents protocol, running the iTRAQ? Reagents protocol, and preparing the sample mixture for analysis using cationexchange chromatography.

Before You Begin
Required Materials Preparing Your Sample
Table 2-1

See Applied Biosystems iTRAQ? Reagents Chemistry Reference Guide, Chapter 1, “User-Supplied and Kit Materials.” If your sample contains a substance that may interfere with the iTRAQ? Reagents protocol (Table 2-1), perform acetone precipitation to clean up the sample.
When to Perform Acetone Precipitation Before beginning the protocol. If the substance is needed to solubilize your sample, after reducing the protein and blocking cysteine. Before trypsin digestion.

Substances that may interfere with the iTRAQ? Reagents protocol
Potential Interference Interfere with cysteine blocking. Inactivate trypsin.

Potential Interfering Substance Thiols (for example, DTT and mercaptoethanol) High amounts of detergents and denaturants (see Table 2-2 for concentration limits of some acceptable detergents/denaturants.) Active proteases Primary amines (for example, those in: ? ? ? ? ? Ammonium acetate Ammonium bicarbonate Ammonium citrate Ammonium tartrate AMPD [2-amino-2-methyl-1,3propanediol] ? Aminoguanidine bicarbonate salt ? AMP [2-amino-2-methyl-1-propanol] ? Ethanolamine, gly-gly, tris) For information, see the Applied Biosystems iTRAQ? Reagents Chemistry Reference Guide, Appendix A, “Labeling the Protein Digests.”

React with iTRAQ? Reagents, interfering with labeling.

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Before You Begin

If your sample is insoluble after adding Dissolution Buffer and Denaturant (steps 1 and 2 on page 2-5), choose an alternative detergent/denaturant or buffer (Table 2-2). For information, see the Applied Biosystems iTRAQ? Reagents Chemistry Reference Guide, Appendix A, “Dissolving the Sample.”
Table 2-2 Recommended alternative detergent/denaturant and buffers
Alternative Buffer BES BICINE Boric acid CHES DIPSO EPPS HEPBS HEPES HEPPSO MOBS MOPS Phosphate Buffered Saline PIPES POPSO

Alternative Detergent/Denaturant (Concentration Limit at Trypsin Digestion) SDS (0.05%) OG (octyl B-D-glucopyranoside) (0.1%) NP -40 (0.1%) Triton X-100 (0.1%) Tween? 20 (0.1%) CHAPS (0.1%) Urea (<1M)
? ?

Note: When using urea, always use a fresh
solution. When reducing a sample containing urea, incubate the tubes at 37 °C for 1 hour (step 5 on page 2-5).

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Chapter 2 iTRAQ? Reagents Protocol

Acetone Precipitation IMPORTANT! If you perform acetone precipitation after trypsin digestion, sample can be lost.

CHEMICAL HAZARD. Acetone is a flammable liquid and vapor. Exposure may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry the skin. Exposure may cause central nervous system depression. Keep away from heat, sparks, and flame. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. To clean up a sample by acetone precipitation: 1. Chill acetone and the sample tube containing the sample to 4 °C. 2. Add six volumes of cold acetone to the cold sample tube. 3. Invert the tube three times. 4. Incubate the tube at –20 °C until precipitate forms (30 minutes to four hours). 5. Decant the acetone. Do not dry. 6. Use the precipitated pellet as your sample in “Reducing the Proteins and Blocking Cysteine,” step 1, page 2-5.

Testing the Protocol

If you are running the protocol for the first time, Applied Biosystems recommends that you run your Control sample through the entire protocol before you run an actual experiment. If you lack enough control sample to test the protocol, prepare a sample using your sample conditions and the Six-Protein Mix provided in the Methods Development Kit. Label the sample with one of the iTRAQ? Reagents. Successful analysis of the iTRAQ? Reagent-labeled Control sample verifies that your sample preparation protocol does not interfere with digestion and iTRAQ? Reagents labeling. If the analysis fails, modify your sample preparation or the protocol according to the guidelines in the Applied Biosystems iTRAQ? Reagents Chemistry Reference Guide, Appendix A, “Modifying the iTRAQ? Reagents Protocol.”

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Running the Protocol

Running the Protocol
Running the iTRAQ? Reagents protocol involves: ? Reducing the sample, denaturing the sample, and blocking the cysteines ? Digesting the proteins with trypsin ? Labeling the digested proteins with iTRAQ? Reagents ? Combining the labeled digests into one sample mixture

Reducing the Proteins and Blocking Cysteine
CHEMICAL HAZARD. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Denaturant (2% SDS) causes eye and skin irritation. Reducing Reagent causes eye, skin, and respiratory tract irritation. Cysteine Blocking Reagent is a flammable liquid and vapor. Exposure causes eye and respiratory tract irritation and central nervous system depression.
IMPORTANT! If your sample contains thiols, perform acetone precipitation (see “Preparing Your Sample” on page 2-2).

1. To each of up to four sample tubes containing 5 to 100 ?g of sample (or the precipitated pellet from acetone precipitation), add 20 ?L Dissolution Buffer. 2. Add 1 ?L of the Denaturant in the kit and vortex to mix. If the sample does not dissolve, see the Applied Biosystems iTRAQ? Reagents Chemistry Reference Guide, Appendix A, “Dissolving the Sample.” 3. To each sample tube, add 2 ?L Reducing Reagent. 4. Vortex to mix, then spin. 5. Incubate the tubes at 60 °C for 1 hour. 6. Spin to bring the sample to the bottom of the tube. 7. To each tube, add 1 ?L Cysteine Blocking Reagent. 8. Vortex to mix, then spin. 9. Incubate the tubes at room temperature for 10 minutes.
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Chapter 2 iTRAQ? Reagents Protocol

Digesting the Proteins with Trypsin
CHEMICAL HAZARD. Trypsin causes eye, skin, and respiratory tract irritation. Exposure may cause an allergic reaction. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
IMPORTANT! If necessary, perform acetone precipitation to remove contaminants that may interfere with trypsin digestion or iTRAQ? Reagents labeling (see “Preparing Your Sample” on page 2-2).

1. Reconstitute a vial of trypsin with 25 ?L of Milli-Q? water or equivalent. (If preparing three to four samples, reconstitute two vials.) 2. Vortex to mix, then spin. 3. To each sample tube, add 10 ?L of the trypsin solution. 4. Vortex to mix, then spin. 5. Incubate the tubes at 37 °C overnight (12 to 16 hours). 6. Spin to bring the sample digest to the bottom of the tube.
Note: In order to maximize labeling efficiency, the volume of the

sample digest must be less than 50 ?L. If the volume of the sample digest is greater than 50 ?L, dry the sample in a centrifugal vacuum concentrator, then reconstitute with 30 ?L Dissolution Buffer.

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Running the Protocol

Labeling the Protein Digests with the iTRAQ? Reagents
CHEMICAL HAZARD. Read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Ethanol is a flammable liquid and vapor. Exposure causes eye, skin, and respiratory tract irritation and may cause central nervous system depression and liver damage. iTRAQ? Reagents 114-117 cause eye and respiratory tract irritation.
Exposure may cause blood damage.

1. Allow each vial of iTRAQ? Reagent required to reach room temperature. 2. Spin to bring the solution to the bottom of the tube. 3. Add 70 ?L of ethanol to each room-temperature iTRAQ? Reagent vial. 4. Vortex each vial to mix, then spin. 5. Transfer the contents of one iTRAQ? Reagent vial to one sample tube. For example, for a duplex-type experiment, transfer the contents of the iTRAQ? Reagent 114 vial to the sample 1 protein digest tube and transfer the contents of the iTRAQ? Reagent 117 vial to the sample 2 protein digest tube. 6. Vortex each tube to mix, then spin. 7. Incubate the tubes at room temperature for 1 hour.

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Chapter 2 iTRAQ? Reagents Protocol

Combining the iTRAQ? Reagent-Labeled Digest Samples
CHEMICAL HAZARD. iTRAQ? Reagents
114-117 cause eye and respiratory tract irritation. Exposure may cause blood damage. Read the MSDS, and follow the handling instructions.

Wear appropriate protective eyewear, clothing, and gloves. 1. Combine the contents of each iTRAQ? Reagent-labeled sample tube into one tube. a 2. Vortex to mix, then spin.

a. (Optional) Before combining the samples, you can analyze an aliquot of each sample by MS/MS to confirm the presence of iTRAQ? Reagentlabeled peptides. Before analyzing, reduce the organic concentration, then clean up the sample using a ZipTip?. In MS/MS analysis, verify that you see peaks at the m/z of the appropriate iTRAQ? Reagent reporter group. If not, relabel the protein digest.
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Preparing the Sample Mixture for LC/MS/MS Analysis

Preparing the Sample Mixture for LC/MS/MS Analysis
The following substances in an iTRAQ? Reagent-labeled sample mixture may interfere with LC/MS/MS analysis: ? Dissolution Buffer ? 75% organic solvent (ethanol and acetonitrile) ? 1 mM Reducing Reagent (tris-(2-carboxyethyl) phosphine [TCEP]) ? 0.02% SDS ? 5 mM calcium chloride ? Excess iTRAQ? Reagents Before you perform LC/MS/MS analysis, clean up the sample mixture using cation exchange chromatography. For a simple sample mixture, use the cation exchange system provided in the Methods Development Kit (first order only). See the procedure below. For a complex mixture, use high-resolution cation exchange chromatography (see the Applied Biosystems iTRAQ? Reagents Chemistry Reference Guide, Appendix C, “High-Resolution Cation Exchange Chromatography”.)

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Chapter 2 iTRAQ? Reagents Protocol

Preparing a Simple Sample Mixture for LC/MS/MS Analysis
This section describes using the cation exchange system in the Methods Development Kit (first order only). For information about required materials and assembling the cartridge, see Applied Biosystems iTRAQ? Reagents Chemistry Reference Guide, Appendix C, “Required Materials” and “Before You Begin”. CHEMICAL HAZARD. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Cation Exchange Buffer–Load, Cation Exchange Buffer–Elute, Cation Exchange Buffer–Clean, and Cation Exchange Buffer–Storage contain acetonitrile, a flammable liquid and vapor. Exposure causes eye, skin, and respiratory tract irritation and may cause blood damage. Keep away from heat, sparks, and flame. iTRAQ? Reagents 114-117 cause eye and respiratory tract irritation. Exposure may cause blood damage. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

Performing Cation-Exchange Chromatography

1. Reduce the concentrations of buffer salts and organics by diluting the sample mixture at least 10 fold with Cation Exchange Buffer-Load. 2. Vortex to mix. 3. Check the pH using pH paper. If the pH is not between 2.5 and 3.3, adjust by adding more Cation Exchange Buffer–Load. 4. To condition the cartridge, inject 1 mL of the Cation Exchange Buffer–Clean. Divert to waste. 5. Inject 2 mL of the Cation Exchange Buffer–Load. Divert to waste. 6. Slowly inject (≈1 drop/second) the diluted sample mixture onto the cation-exchange cartridge and collect the flow-through in a sample tube.

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Preparing the Sample Mixture for LC/MS/MS Analysis

7. Inject 1 mL of the Cation Exchange Buffer–Load to wash the TCEP, SDS, calcium chloride, and excess iTRAQ? Reagents from the cartridge. Collect the flow-through in the same sample tube used in step 6. (Keep the flow-through until you verify by MS/MS analysis that loading on the cation-exchange cartridge was successful. If loading fails, you can repeat loading using the flow-through after you troubleshoot the cause of the loading failure.) 8. To elute the peptides, slowly inject (~1 drop/second) 500 ?L of the Cation Exchange Buffer–Elute. Capture the eluate in a fresh 1.5-mL tube. Collect the eluted peptides as a single fraction. 9. Wash the undigested proteins such as trypsin from the cationexchange cartridge by injecting 1 mL of the Cation Exchange Buffer–Clean. Divert to waste. 10. If you have additional sample mixtures, repeat steps 5 through 8. If you do not have additional sample mixtures, store the cartridge as described below.

Storing the Cation-Exchange Cartridge

1. After cleaning the cartridge (step 9 above), inject 2 mL of the Cation Exchange Buffer–Storage. 2. Remove the cartridge, then seal the ends of the cartridge with the two end caps. 3. Record the number of times the cartridge has been used. 4. Store the cartridge at 2 to 8 °C. 5. Clean the needle-port adapter, outlet connector, and syringe with water.

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Chapter 2 iTRAQ? Reagents Protocol

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Notes

iTRAQ? Reagents Protocol

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Notes-1

Notes

Notes-2

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Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: +1 650.638.5800 Toll Free (In North America): +1 800.345.5224 Fax: +1 650.638.5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For international office locations, please call our headquarters or see our Web site at www.appliedbiosystems.com. Technical Support In North America, call +1 800.899.5858. Outside North America, see our Web site at www.appliedbiosystems.com.

Applera is committed to providing the world’s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Printed in USA, 10/2004 Part Number 4350831 Rev. C


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