ICAT™ Kit for Protein Labeling
(Monoplex Version)
Protocol for Modifying Proteins with an
Isotope-Labeled, Sulfhydryl-Modifying
Biotinylation Reagent
Operating Instructions
1
Product Description
1.
The ICAT™ Kit for Protein Labeling (Monoplex Version) facilitates
quantitative analysis of proteins. The biotinylated peptides, products of
the reactions generated by this kit, can be separated by capillary
reversed-phase HPLC and analyzed by mass spectrometry. This
monoplex kit lets you label a control and a test protein sample for up to
three assays (if you purchase the 3-assay starter kit), or up to 10
assays (if you purchase the 10-assay kit).
3.
Remove nonderivitized
material
The ICAT Kit for Protein Labeling complements 2D gel electrophoresis
techniques by allowing for quantitation of a wide range of proteins,
including membrane and low-abundance proteins.
8.
Protocol Overview
Separate ICAT
Reagent-labeled
peptides by capillary
reversed-phase HPLC
Each ICAT Reagent consists of three moieties:
A sulfhydryl-specific reactive group (iodoacetamide) to
derivitize cysteine residues
•
An affinity ligand (biotin) that acts as a specific capture “handle”
•
A linker region that contains either 8 deuterium atoms (D8) or
0 deuterium atoms (D0)
5.
Load the eluted
peptides on the
affinity column
Clean up
samples using
cation exchange
6.
Benefits
Label samples
with ICAT
Reagents
4.
Digest the protein
samples with
trypsin
The protein analysis approach described in this kit requires that you
provide protein samples and various laboratory materials. Refer to
“User-Supplied Materials and Equipment” on page 4 for details.
•
2.
Denature and
reduce the
protein samples
7.
Elute derivitized
peptides
9.
Identify and quantify by
electrospray, MALDI, or
both electrospray and
MALDI
Figure 1 Steps in the Protocol
The protocol in this document is based on the addition of ICAT
Reagents to two different protein samples. One reagent, D0, is added
to a control sample; the second reagent, D8 (Figure 2), is added to a
test sample.
After ICAT Reagent is added to the control and test samples, samples
are derivitized, combined, then digested with trypsin. Excess reagent
and trypsin are removed using cation exchange chromatography.
Biotinylated, cysteine-containing tryptic fragments are captured on an
avidin affinity column, and labeled peptides are separated from nonlabeled material. After elution from the avidin column, labeled peptides
are further separated using capillary reversed-phase HPLC, then
analyzed by mass spectrometry. Relative quantitation between the
control and test samples is determined from the mass peak ratio of D8labeled peptide to D0-labeled peptide. Protein identification is
performed by MS/MS.
Contents
Page
Product Description .............................................................. 1
Materials ............................................................................... 2
Safety.................................................................................... 4
Testing the Protocol .............................................................. 4
Protein Labeling and Tryptic Digestion ................................. 5
Purifying the Biotinylated Peptides ....................................... 7
Analyzing the Fractions and Peptides................................... 8
Technical Support ............................................................... 10
Ordering Information ............................................................11
References...........................................................................11
For additional information on the analysis of proteins using ICAT
Reagents, refer to Section 10, "References."
Figure 1 outlines the steps in this protocol.
1
Laminin Peptide and ICAT Reagent Reference Information
Mass and chemical information relevant to the Laminin Peptide
Standard and the ICAT Reagent is provided below.
Laminin:
•
•
•
•
•
•
•
•
Sequence: Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg
Composition: C40H62N12O14S
Average molecular weight: 967.1
Monoisotopic mass: 966.4229
Monoisotopic MH+: 967.4307
CAS: 110590-60-8
Monoisotopic MH+ after D0 dervitization: 1409.6552
Monoisotopic MH+ after D8 dervitization: 1417.7054
Item
Volume/Qty.
Description
Laminin
Peptide
Standard
2 vials
Test peptide for the kit.
Trypsin (TPCKtreated)
3 vials
Cleaves peptide bonds on the
carboxyl side of lysine and
arginine residues.
Denaturing
Buffer
(pH 8.5)
1 vial,
1.5 mL/vial
Disrupts the hydrogen,
hydrophobic, and electrostatic
bonds of the proteins. Contains
50 mM Tris and 0.1% SDS.
Reducing
Reagent
1 vial,
100 µL/vial
Reduces the disulfide bonds of
the proteins. Contains 50 mM
TCEP.
Dissolution
Buffer
(pH 8.5)
1 vial,
1.5 mL/vial
Dissolves the laminin in the test
phase of the protocol. Contains
50 mM Tris.
ICAT Cartridge
–Cation
Exchange
one 200-µL
cartridge
Contains POROS® 50 HS,
50-µm particle size, 4.0 mm ×
1.5 cm. Identified by a white
band.
Cation
Exchange
Buffer–Load
(pH 3.0)
100 mL
Phosphate buffer with acetonitrile
that adjusts the pH and lowers
the salt concentration.
Cation
Exchange
Buffer–Elute
(pH 3.0)
100 mL
Phosphate buffer with acetonitrile
and salt that raises the salt
concentration to elute the
peptides.
Cation
Exchange
Buffer–Clean
(pH 3.0)
100 mL
Phosphate buffer with acetonitrile
and high salt concentration that
cleans the cation exchange
cartridge after peptide elution.
Cation
Exchange
Buffer–Storage
(pH 3.0)
100 mL
Phosphate buffer with acetonitrile
and sodium azide that maintains
the proper pH and prevents
growth of microorganisms.
ICAT Cartridge–
Avidin
one 200-µL
cartridge
Purifies biotinylated molecules.
Can be regenerated and reused
for up to 10 assays. (4.0 mm ×
1.5 cm; identified by a black
band)
Affinity Buffer–
Elute
100 mL
Conditions the affinity cartridge
and elutes ICAT Reagent-labeled
peptides. Contains 0.4%
trifluoroacetic acid and 30.0%
acetonitrile.
Affinity Buffer–
Load (pH 7.2)
100 mL
Phosphate buffer that adjusts the
pH to approximately 7.2.
Affinity Buffer–
Wash 1
(pH 7.2)
100 mL
Phosphate buffer that decreases
the salt concentration.
Affinity Buffer–
Wash 2
(pH 8.3)
100 mL
Bicarbonate solution with
methanol that decreases the salt
concentration and reduces
nonspecifically bound peptides.
Affinity Buffer–
Storage
(pH 7.2)
100 mL
Phosphate buffer with sodium
azide that maintains the proper
pH and prevents growth of
microorganisms.
Cartridge
holder
(available only
in Starter Kit)
1
(for 200-µL
cartridges)
Reusable bayonet-style holder
for 200-µL cation exchange and
avidin affinity cartridges.
Needle-port
adapter
(available only
in Starter Kit)
1
Provides a secure connection for
the HPLC syringe needle (while
injecting onto the column).
ICAT Reagent:
•
•
•
•
•
•
•
•
•
Composition: C20H35N4O5SI
Average molecular weight (D0): 570.5
Monoisotopic mass (D0): 570.1373
Monoisotopic MH+ (D0): 571.1446
Average molecular weight (D8): 578.5
Monoisotopic mass (D8): 578.1875
Monoisotopic MH+ (D8): 579.1948
Monoisotopic mass added to peptide (D0 reagent): 442.2250
Monoisotopic mass added to peptide (D8 reagent): 450.2752
Figure 2 shows the structure of ICAT Reagent D8.
O
NH
HN
H
N
D D
S
O
D D
O
D D
O
O
D D
I
N
H
Figure 2 ICAT Reagent D8 Structure
2
Materials
This section describes:
•
•
•
2.1
Materials in the kits
Storage conditions
User-supplied materials
Materials in the Kits
This section describes the materials provided in the:
•
•
3-Assay ICAT Starter Kit (includes hardware required for the
protocol)
10-Assay ICAT Kit (does not include required hardware)
Materials in the 3-Assay ICAT Starter Kit
The following table lists the materials in the 3-Assay ICAT Starter Kit.
Caution: When you receive shipping container #1 of 2, immediately
remove the 3-Assay Reagent box from the container and store it at –15
to –25 °C. Store items in shipping container #2 of 2 at 2 to 8 °C.
Item
Volume/Qty.
Description
ICAT Reagent
D8
4 vials,
1 unit/viala
Sulfhydryl-modifying biotinylation
heavy reagent, typically used to
label the test sample.
ICAT Reagent
D0
4 vials,
1 unit/viala
Sulfhydryl-modifying biotinylation
light reagent (hydrogen), typically
used to label the control sample.
2
Item
Volume/Qty.
Description
Item
Volume/Qty.
Description
Outlet tubing kit
(available only
in Starter Kit)
1
1/16-inch O.D. PEEK™ tubing
and 10-32 compression screw for
connecting to the outlet side of
the cartridge holder.
Cation
Exchange
Buffer–Storage
(pH 3.0)
100 mL
ICAT Kit for
Protein
Labeling
Operating
Instructions
1
This document.
Phosphate buffer with
acetonitrile and sodium azide
that maintains the proper pH
and prevents growth of
microorganisms.
ICAT
Cartridge–
Avidin
one 200-µL
cartridge
ICAT Kit for
Protein
Labeling Quick
Card
1
Purifies biotinylated molecules.
Can be regenerated and reused
for up to 10 assays. (4.0 mm ×
1.5 cm; identified by a black
band)
Affinity Buffer–
Elute
100 mL
Conditions the affinity cartridge
and elutes ICAT Reagentlabeled peptides. Contains 0.4%
trifluoroacetic acid and 30.0%
acetonitrile.
Affinity Buffer–
Load
(pH 7.2)
100 mL
Phosphate buffer that adjusts the
pH to approximately 7.2.
Affinity Buffer–
Wash 1
(pH 7.2)
100 mL
Phosphate buffer that decreases
the salt concentration.
Affinity Buffer–
Wash 2
(pH 8.3)
100 mL
Bicarbonate solution with
methanol that decreases the salt
concentration and reduces
nonspecifically bound peptides.
Affinity Buffer–
Storage
(pH 7.2)
100 mL
Phosphate buffer with sodium
azide that maintains the proper
pH and prevents growth of
microorganisms.
ICAT Kit for
Protein
Labeling
Operating
Instructions
1
This document.
ICAT Kit for
Protein
Labeling Quick
Card
1
Laminated card that provides a
quick reference to the steps in
this protocol.
Laminated card that provides a
quick reference to the steps in
this protocol.
a. One unit of reagent labels 50 µg (approximately 50 nmol) of laminin.
Materials in the 10-Assay ICAT Kit
The following table lists the materials in the 10-Assay ICAT Kit.
Caution: When you receive shipping container #1 of 2, immediately
remove the 10-Assay Reagent box from the container and store it at
–15 to –25 °C. Store items in shipping container #2 of 2 at 2 to 8 °C.
Item
Volume/Qty.
Description
ICAT Reagent
D8
11 vials,
1 unit/viala
Sulfhydryl-modifying biotinylation
heavy reagent, typically used to
label the test sample.
ICAT Reagent
D0
11 vials,
1 unit/viala
Sulfhydryl-modifying biotinylation
light reagent (hydrogen),
typically used to label the control
sample.
Laminin
Peptide
Standard
2 vials
Test peptide for the kit.
Trypsin (TPCKtreated)
10 vials
Cleaves peptide bonds on the
carboxyl side of lysine and
arginine residues.
Denaturing
Buffer
(pH 8.5)
1 vial,
1.5 mL/vial
Disrupts the hydrogen,
hydrophobic, and electrostatic
bonds of the proteins. Contains
50 mM Tris and 0.1% SDS.
Reducing
Reagent
1 vial,
100 µL/vial
Reduces the disulfide bonds of
the proteins. Contains 50 mM
TCEP.
Dissolution
Buffer
(pH 8.5)
1 vial,
1.5 mL/vial
Dissolves the laminin in the test
phase of the protocol. Contains
50 mM Tris.
ICAT Cartridge
–Cation
Exchange
one 200-µL
cartridge
Cation
Exchange
Buffer–Load
(pH 3.0)
100 mL
Cation
Exchange
Buffer–Elute
(pH 3.0)
100 mL
Cation
Exchange
Buffer–Clean
(pH 3.0)
100 mL
a. One unit of reagent labels 50 µg (approximately 50 nmol) of laminin.
2.2
Contains POROS® 50 HS,
50-µm particle size, 4.0 mm ×
1.5 cm. Identified by a white
band.
Phosphate buffer with
acetonitrile that adjusts the pH
and lowers the salt
concentration.
Phosphate buffer with
acetonitrile and salt that raises
the salt concentration to elute
the peptides.
Phosphate buffer with
acetonitrile and high salt
concentration that cleans the
cation exchange cartridge after
peptide elution.
3
Storage Conditions
Item
Storage Conditions (°C)
ICAT Reagent D8
–15 to –25
ICAT Reagent D0
–15 to –25
Laminin Peptide Standard
–15 to –25
Trypsin (TPCK-treated)
–15 to –25
Denaturing Buffer
–15 to –25
Reducing Reagent
–15 to –25
Dissolution Buffer
–15 to –25
ICAT Cartridge–Cation Exchange
2 to 8
Cation Exchange Buffer–Load
2 to 8
Cation Exchange Buffer–Elute
2 to 8
Cation Exchange Buffer–Clean
2 to 8
Cation Exchange Buffer–Storage
2 to 8
ICAT Cartridge–Avidin
2 to 8
Affinity Buffer–Elute
2 to 8
Affinity Buffer–Load
2 to 8
Item
Storage Conditions (°C)
Affinity Buffer–Wash 1
2 to 8
Affinity Buffer–Wash 2
2 to 8
Affinity Buffer–Storage
2 to 8
2.3
3.2
Ordering MSDSs
You can obtain free copies of MSDSs for chemicals distributed by
Applied Biosystems using the contact information below. For chemicals
not distributed by Applied Biosystems, call the chemical manufacturer.
To order
MSDSs ...
User-Supplied Materials and Equipment
Over the Internet
Then ...
1. Go to www.appliedbiosystems.com/
techsupp.
Item
~ Volume or
Quantity per
Assay
Disposable gloves
As needed
Pipettors and tips suitable for 1- to 1000-µL
volumes
As needed
Aluminum foil
As needed
Syringe (2-inch blunt needle, 18 gauge, 2.5 mL
capacity)
1
Fraction collection tubes
As needed
Control sample
100 µg
Test sample
100 µg
HPLC-grade water
50 mL
Heating block
1
Bench-top centrifuge
1
Vortex
1
1. From the U.S. or Canada, dial
1.800.487.6809, or from outside the U.S.
and Canada, dial 1.858.712.0317.
Mass spectrometer
1
2. Follow the voice instructions.
Capillary reversed-phase HPLC system
1
New Objective, Inc. coated fused-silica spray tip
(Cat. #FS360-20-10-CE-20)a
1
4
Tubing fitting from LC Packings
(Cat. #TF-250/350)a
1
This section describes:
2. Under the Resource Libraries heading,
click MSDSs
• If you have the product part number or
the keyword(s), select Click Here, enter
the part number or keyword(s) in the
appropriate field, then click Search.
• If you have the MSDS document
number or the Document on Demand
index number, enter one of these
numbers in the appropriate field, then
click Search.
3. Open or download a PDF version (using
Adobe® Acrobat® Reader™) of the
document by selecting it, or choose to
have the document sent to you by fax or
email.
By telephone
Testing the Protocol
•
•
a. Required only if using a Protana NanoES source on an Applied
Biosystems/MDS SCIEX QSTAR™ Hybrid LC/MS/MS Quadrupole TOF
System. See “Choosing a Source for the QSTAR System” on page 8.
4.1
3
Safety
3.1
Chemical hazard cautions and warnings
Ordering MSDSs
Chemical Hazard Cautions and Warnings
To test the kit components, Applied Biosystems recommends that you
first run the protocol with the Laminin Peptide Standard supplied in this
kit. Testing involves derivitizing the laminin with ICAT Reagents D0 and
D8, then analyzing by mass spectrometry.
WARNING: Some of the chemicals used with this protocol are
potentially hazardous and can cause injury, illness, or death.
•
•
•
•
•
Testing with the Laminin Peptide Standard
WARNING: CHEMICAL HAZARD. ICAT Reagents D0 and D8 are
harmful if inhaled, absorbed through the skin, or swallowed. Exposure
causes eye, skin, and respiratory tract irritation. ICAT Reagents D0 and
D8 may cause allergic reactions and are considered possible
birth-defect hazards. Please read the MSDS and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and
gloves.
This section describes:
•
•
Testing the kit components with the laminin peptide standard
Testing the protocol with a known protein
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.
Minimize contact with and inhalation of chemicals. Wear
appropriate personal protective equipment when handling
chemicals (e.g., safety glasses, gloves, or protective clothing).
For additional safety guidelines, consult the MSDS.
Do not leave chemical containers open. Use chemicals only
with adequate ventilation.
Check regularly for chemical leaks or spills. If a leak or spill
occurs, follow the recommended cleanup procedures in the
MSDS.
Comply with all local, state/provincial, or national laws and
regulations related to chemical storage, handling, and disposal.
Note: Because ICAT Reagents D0 and D8 in the Dissolution Buffer
degrade approximately 10% every 24 hours at room temperature,
Applied Biosystems recommends that you use the reagents as soon as
possible after reconstitution.
1. Resuspend one vial of the Laminin Peptide Standard in 100 µL of
the Dissolution Buffer.
2. Repeat step 1 using the second vial of Laminin Peptide Standard.
3. Add 2 µL of the Reducing Reagent to each of the laminin vials.
4. Boil both vials for 10 minutes, then cool.
5. Spin both vials to force all solution to the bottom of the vials.
4
6. For control samples, remove from each vial a 1-µL aliquot for MS
analysis. For MALDI analysis, mix each aliquot with an appropriate
matrix (for example, alpha-cyano-4-hydroxycinnamic acid) in a
1:1 ratio (v/v).
2. Using the two tubes that contain your known sample, follow the
steps in Section 5.1, Section 5.2, and Section 5.3.
3. Complete step 1 in Section 5.4. Then, in place of steps 2 through 4,
do the following:
7. From one of the laminin vials, transfer the remaining 99 µL of the
laminin solution to an ICAT Reagent D0 vial.
a. In one tube, mix a 50-µL aliquot of the D0-labeled sample with a
50-µL aliquot of the D8-labeled sample. (This is your 1:1
sample.)
8. From the other laminin vial, transfer the remaining 99 µL of the
laminin solution to an ICAT Reagent D8 vial.
b. In a second tube, mix a 25-µL aliquot of the D0-labeled sample
with a 50-µL aliquot of the D8-labeled sample. (This is your
1:2 sample.)
9. Vortex the vials, cover the vials with foil, then incubate in the dark
for 1 hour at room temperature.
c. Dissolve a vial of trypsin in 200 µL of HPLC-grade water.
10. Spin both vials to force all solution to the bottom of the vials.
d. Add half of the trypsin solution to each sample tube.
11. Combine both vials and mix.
4. Perform the rest of the protocol (from step 5 in Section 5.4) with the
1:1 and 1:2 protein mixes.
12. Remove a 1-µL aliquot of the laminin-ICAT Reagent solution for
MS analysis (test sample). For MALDI analysis, mix the aliquot
with an appropriate matrix (for example, alpha-cyano-4hydroxycinnamic acid) in a 1:1 ratio (v/v).
This test allows you to qualify the protocol with a sample more complex
than the laminin peptide, and to verify that you can use the protocol to
quantitate to within 20 to 30% of the expected values.
Figure 3 shows representative mass spectra (from a Voyager-DE™
PRO Biospectrometry™ Workstation) of D0- and D8-labeled laminin.
The ICAT Reagent-modified peptide has a mass of 1409.7 (D0) and
1417.7 (D8). The excess ICAT Reagent reacts with the reducing
reagent (TCEP), resulting in the mass pair at 693.6 and 701.7.
Although your spectrum may not show the signals for the reaction
products of the ICAT dimer with TCEP and laminin (at 1135.7, 1151.8,
1852.6, and 1867.7), they are shown in Figure 3 for reference.
5
Protein Labeling and Tryptic Digestion
This section describes:
•
•
•
•
•
Sample preparation
Denaturing and reducing
Starting the ICAT Reagent reaction
Digesting the proteins with trypsin
Cleaning up the peptides using cation exchange
701.7
1151.8
1135.7
1867.7
1852.6
5.2
Denaturing and Reducing the Proteins
CAUTION: CHEMICAL HAZARD. Reducing Reagent may cause
eye, skin, and respiratory tract irritation. Please read the MSDS before
handling these products and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves.
Figure 3 Spectrum for D8- and D0-Labeled Laminin
4.2
Sample Preparation
Make sure your samples are dry or concentrated and do not contain
high acid or salt concentrations or detergent. Low pH can adversely
affect the pH-dependent reaction of the sulfhydryl groups with the ICAT
Reagent. High salt concentrations can result in peptides not binding to
the cation exchange column. If necessary, clean up the samples using
chromatography, dialysis, acetone precipitation, or ultracentrifugation
before proceeding.
ICAT dimer and Laminin
1417.7
1409.7
ICAT and Laminin
ICAT dimer and TCEP
ICAT and TCEP
5.1
693.6
1. Add 100 µL of the Denaturing Buffer to a tube containing 100 µg of
the control protein sample. (If working with a concentrated sample
solution, add Denaturing Buffer to bring the volume up to 100 µL.)
Testing with a Known Protein
2. Add 100 µL of the Denaturing Buffer to a tube containing 100 µg of
the test protein sample. (If working with a concentrated sample
solution, add Denaturing Buffer to bring the volume up to 100 µL.)
WARNING: CHEMICAL HAZARD. ICAT Reagents D0 and D8 are
harmful if inhaled, absorbed through the skin, or swallowed. Exposure
causes eye, skin, and respiratory tract irritation. ICAT Reagents D0 and
D8 may cause allergic reactions and are considered possible birthdefect hazards. Please read the MSDS and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and
gloves.
3. Add 2 µL of the Reducing Reagent to both the control and test
sample tubes.
4. Boil each sample for 10 minutes, then allow the samples to cool.
5.3
Before you run your complex samples for the first time, Applied
Biosystems strongly recommends that you perform this protocol with a
well-characterized protein that contains multiple cysteines (e.g., 25 to
50 µg of bovine serum albumin or 100 µg of bovine lactalbumin).
Starting the ICAT Reagent Reaction
WARNING: CHEMICAL HAZARD. ICAT Reagents D0 and D8 are
harmful if inhaled, absorbed through the skin, or swallowed. Exposure
causes eye, skin, and respiratory tract irritation. ICAT Reagents D0 and
D8 may cause allergic reactions and are considered possible birthdefect hazards. Please read the MSDSs and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and
gloves.
1. Into each of two tubes, add equal amounts of your known protein
sample (either a lyophilized sample at a known concentration or a
sample in a concentrated stock solution).
5
1. Spin the control and test samples to force all solution to the bottom
of the tubes.
2. Transfer the contents of the control sample to a vial of the D0
reagent.
3. Transfer the contents of the test sample to a vial of the D8 reagent.
4. Mix each sample vial thoroughly.
5. Wrap each sample vial in aluminum foil and incubate for 1 hour at
room temperature.
5.4
Syringe
Digesting the Proteins with Trypsin
WARNING! CHEMICAL HAZARD. Trypsin may cause eye, skin, and
respiratory tract irritation. Exposure may also cause an allergic
reaction. Please read the MSDS and follow the handling instructions.
Wear appropriate protective eyewear, clothing, and gloves.
Syringe needle
1. Spin both vials to force all solution to the bottom of the vials.
Needle-port
adapter
2. Into one tube, combine a 95-µL aliquot of the control sample and a
95-µL aliquot of the test sample. Mix thoroughly.
3. Dissolve a vial of trypsin in 200 µL of HPLC-grade water.
4. Add the trypsin solution to the sample mixture, then mix thoroughly.
5. Incubate overnight (or 12 to 16 hours) at 37 °C.
5.5
Cleaning Up the Peptides Using Cation
Exchange
Column holder
with cartridge
General Injection Guidelines
•
•
•
•
Fill a clean syringe with the appropriate solution.
Insert the syringe needle into the needle-port adapter and
tighten the adapter.
After each injection, wash the needle and syringe several times
with water and once with the next solution before refilling the
syringe for the next injection.
For washing and conditioning steps, inject solution so that 2 to
3 drops/second flow from the outlet. For eluting and loading
steps, inject solution so that approximately 1 drop/second flows
from the outlet.
Compression screw
1/16-inch O.D. PEEK
outlet tubing
Figure 4 Column Connection
Loading Sample on the Cation Exchange Column
Preparing the Cation Exchange Column
WARNING: CHEMICAL HAZARD. The Cation Exchange Buffer–
Load and Cation Exchange Buffer–Elute contain acetonitrile, a
flammable liquid and vapor. Exposure may cause eye, skin, and
respiratory tract irritation, central nervous system depression, and
heart, liver, and kidney damage. Please read the MSDS and follow the
handling instructions. Wear appropriate protective eyewear, clothing,
and gloves.
WARNING: CHEMICAL HAZARD. The Cation Exchange Buffer–
Load contains acetonitrile, a flammable liquid and vapor. Exposure
may cause eye, skin, and respiratory tract irritation, central nervous
system depression, and heart, liver, and kidney damage. Please read
the MSDS and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves.
1. Assemble the reusable column holder.
1. Transfer the sample mixture (from Section 5.4, step 5) to a tube
with capacity greater than 3 mL.
2. Slide the PEEK tubing into a 10-32 compression screw, then
finger-tighten the compression screw into the outlet end of the
column (see Figure 4).
2. Dilute the sample mixture by adding 2000 µL of the Cation
Exchange Buffer–Load.
3. Connect the needle-port adapter to the inlet end of the column.
3. Slowly inject (~1 drop/second) the diluted sample mixture onto the
cation exchange column and collect the flow-through.
4. Unscrew the bayonet mount to open the column holder, insert the
cation exchange cartridge (in either direction), then close the
holder.
4. Inject 1000 µL of the Cation Exchange Buffer–Load to wash the
TCEP, SDS, and excess ICAT Reagents from the column.
5. To condition the column, inject 2000 µL of the Cation Exchange
Buffer–Load.
5. To elute the peptides, slowly inject (~1 drop/second) 500 µL of the
Cation Exchange Buffer–Elute. Collect the eluted peptides as a
single fraction.
6
Cleaning and Storing the Cation Exchange Column
6.3
WARNING: CHEMICAL HAZARD. The Cation Exchange Buffer–
Clean, Cation Exchange Buffer–Load, and Cation Exchange
Buffer–Storage contain acetonitrile, a flammable liquid and vapor.
Exposure may cause eye, skin, and respiratory tract irritation, central
nervous system depression, and heart, liver, and kidney damage.
Please read the MSDSs and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves.
WARNING: CHEMICAL HAZARD. Affinity Buffer–Wash 2 contains
methanol, a flammable liquid and vapor. Exposure may cause eye,
skin, and respiratory tract irritation, central nervous system depression,
and blindness. Please read the MSDS and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and
gloves.
1. Inject 500 µL of Affinity Buffer–Load and continue to collect (using
the same collection tube).
1. Wash the trypsin from the cation exchange cartridge by injecting
1000 µL of the Cation Exchange Buffer–Clean.
2. Inject 1000 µL of Affinity Buffer–Wash 1, sending the output to
waste. (This step reduces the salt concentration.)
2. If you have additional protein samples, repeat the steps in
Section 5.5 for each sample. (Start with step 5 in "Preparing the
Cation Exchange Column" on page 6.)
3. To remove non-specifically bound peptides and lower the salt
concentration, inject 1000 µL of Affinity Buffer–Wash 2. Collect the
first 500 µL in a new collection tube; send the remaining 500 µL to
waste.
3. To store the cartridge:
a. Overnight, inject 2000 µL of the Cation Exchange Buffer–Load.
More than one day, inject 2000 µL of the Cation Exchange
Buffer–Storage.
6.4
b. Open the bayonet mount of the column holder and remove the
cation exchange cartridge.
Purifying the Biotinylated Peptides
1. To elute the peptides, slowly inject (~1 drop/second) 800 µL of the
Affinity Buffer–Elute.
This section describes:
•
•
•
•
•
Activating the avidin affinity column
Loading sample on the avidin affinity column
Removing non-labeled material
Eluting ICAT Reagent-labeled peptides
Cleaning and storing the avidin affinity column
2. Discard the first 50 µL of eluate.
3. Collect the next 750 µL of eluate in one tube.
6.5
Note: The affinity column has a maximum recommended load of 8
to 10 nmol for a nominal 1-kDa peptide.
6.1
Cleaning and Storing the Avidin Affinity
Column
WARNING: CHEMICAL HAZARD. The Affinity Buffer–Elute
contains acetonitrile, a flammable liquid and vapor. Exposure may
cause eye, skin, and respiratory tract irritation, central nervous system
depression, and heart, liver, and kidney damage. Please read the
MSDS and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves.
Activating the Avidin Affinity Column
WARNING: CHEMICAL HAZARD. The Affinity Buffer–Elute
contains acetonitrile, a flammable liquid and vapor. Exposure may
cause eye, skin, and respiratory tract irritation, central nervous system
depression, and heart, liver, and kidney damage. Please read the
MSDS and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves.
1. Inject 1000 µL of the Affinity Buffer–Elute to clean the cartridge.
2. If you have additional cation exchange fractions, repeat the steps
in Section 6.1 through Section 6.5 for each fraction. (Start with step
2 in Section 6.1.)
1. Insert the avidin affinity cartridge (in either direction) into the
column holder.
3. Neutralize the column by injecting 2000 µL of Affinity Buffer–Load.
4. To store the cartridge:
2. Inject 1000 µL of the Affinity Buffer–Elute. (This step is critical to
the performance of the avidin affinity column.)
a. Overnight, inject 2000 µL of the Affinity Buffer–Load.
More than one day, inject 2000 µL of the Affinity Buffer–
Storage.
3. Inject 2000 µL of the Affinity Buffer–Load.
6.2
Eluting ICAT Reagent-Labeled Peptides
WARNING: CHEMICAL HAZARD. The Affinity Buffer–Elute
contains acetonitrile, a flammable liquid and vapor. Exposure may
cause eye, skin, and respiratory tract irritation, central nervous system
depression, and heart, liver, and kidney damage. Please read the
MSDS and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves.
c. Cap the ends of the cartridge with the two end caps before
storing at 2 to 8 °C.
6
Removing Non-Labeled Material
Loading Sample on the Avidin Affinity Column
b. Open the bayonet mount of the column holder and remove the
cation exchange cartridge.
1. Neutralize the cation exchange fraction by adding 500 µL of the
Affinity Buffer–Load.
c. Cap the ends of the cartridge with the two end caps before
storing at 2 to 8 °C.
2. Place 3 tubes in a rack for collection.
3. Slowly inject (~1 drop/second) the neutralized fraction onto the
column and collect the flow-through.
7
7
Connecting the Capillary Reversed-Phase HPLC System to
the Mass Spectrometry System
Analyzing the Fractions and Peptides
•
•
•
•
•
•
•
7.1
Criteria for additional sample separation
Quantitation notes
Separating by capillary reversed-phase HPLC
Analyzing by electrospray
Analyzing by MALDI
Analyzing by both MALDI and electrospray
ICAT Reagent fragments using MS/MS analysis
•
For complex proteins, Applied Biosystems recommends that
you further separate by capillary reversed-phase HPLC (see
Section 7.3) before analyzing by electrospray or MALDI.
•
For standard proteins and non-complex samples, you may be
able to directly analyze by MALDI or electrospray (without
additional separation by capillary reversed-phase HPLC).
•
For MALDI analysis, connect to a Voyager-DE™ PRO, STR,
RP, or Elite Biospectrometry™ Workstation (via an InterPlate™
Microfraction Collector for MALDI Analysis). For connection
details, refer to the Voyager™ Biospectrometry™ Workstation
User Guide and to the InterPlate™ Microfraction Collector for
MALDI Analysis User Notes.
•
For simultaneous electrospray and MALDI analysis, connect to
an Applied Biosystems/MDS SCIEX QSTAR™ Hybrid LC/MS/
MS Quadrupole TOF System and a Voyager-DE™ PRO, STR,
RP, or Elite Biospectrometry™ Workstation (via an Applied
Biosystems InterPlate™ Microfraction Collector for MALDI
Analysis using a flow splitter). For connection details, refer to
the documentation sources cited in the two previous items.
Go to the appropriate section:
Quantitation Notes
•
Be aware that ICAT Reagent D8 contains 10 to 20% (total) of
D6 and D7 isotopes. Refer to the Certificate of Analysis for
actual amounts.
•
Quantitation for standard proteins or complex samples is
typically within 20 to 30% of expected values.
7.3
For electrospray analysis, connect to an Applied Biosystems/
MDS SCIEX QSTAR™ Hybrid LC/MS/MS Quadrupole TOF
System. For connection details, refer to the QSTAR System
documentation on CD-ROM.
Criteria for Additional Sample Separation
The complexity of your sample determines if you need to further
separate the ICAT Reagent-labeled peptides before analysis:
7.2
•
Note: The suggested settings in the table are based on a capillary
reversed-phase LC system using an LC Packings Ultimate™ Capillary/
Nano LC System with a Switchos™ Micro Column Switching Device.
Column
Name
Flow
Rate
Section 7.6, Analyzing by MALDI and Electrospray
Analyzing by Electrospray
Based on the capillary HPLC column size and its corresponding flow
rate, use the following table to select the ESI source for the QSTAR
System.
Based on the amount of peptide in the sample, use the following table
to select the capillary HPLC column size, flow rate, and injection
volume.
Column
Size
(I.D.)
•
Choosing a Source for the QSTAR System
Choosing the Capillary HPLC Column and Parameters
Injection
Volumeb
(No Preconcen.)
Section 7.5, Analyzing by MALDI
This section provides guidelines and suggestions for electrospray
analysis of the ICAT Reagent-labeled peptides. In this configuration,
the capillary reversed-phase HPLC system is connected to a QSTAR
system.
Before analyzing by electrospray, MALDI, or by both electrospray and
MALDI, perform additional separation of complex peptides by capillary
reversed-phase HPLC.
Injection
Volumea
(Preconcen.)
Section 7.4, Analyzing by Electrospray
•
7.4
Separating by Capillary Reversed-Phase HPLC
Estimated
Amount of
Peptide
Per Sample
•
Column
Size (I.D.)
Flow Rate
ESI Source for QSTAR
300 µm
4 µL/min
IonSpray
180 µm
1 µL/min
MicroIonSpray
75 µm
200 nL/min
Protana NanoESa
a. The Protana NanoES source requires that you fit a New Objective, Inc.
360-µm O.D. coated fused-silica spray tip (Cat. #FS360-20-10-CE-20)
onto the normal tip holder of the source. To connect the New Objective
360-µm O.D. tip to the 280-µm O.D. HPLC tubing, you need a special
Teflon® fitting from LC Packings (Cat. #TF-250/350).
0.2 to 5.0
pmol
300 µm
Capillary300
4 µL/
min
75 µL
1 to 10 µL
0.02 to 1.0
pmol
180 µm
Capillary180
1 µL/
min
50 µL
1 to 5 µL
HPLC Gradient Conditions for QSTAR System Analysis
2 to 500
fmol
75 µm
Nano-75
200 nL/
min
50 µL
1 µL
The following table provides suggested capillary HPLC gradient
conditions for analyzing peptides using a QSTAR system. Note that the
length of the suggested gradient depends on the complexity of the
sample. Samples that have been further fractionated by cation
exchange can use shorter gradients.
a. Assumes that preconcentration is performed with the Switchos device
immediately before injection onto the capillary LC column.
b. Assumes that no preconcentration is performed.
Suggested HPLC Gradient Conditions for Electrospray
8
Mobile phase A
0.1% formic acid in 5% acetonitrile, 95% HPLC-grade
water
Mobile phase B
0.1% formic acid in 95% acetonitrile, 5% HPLC-grade
water
•
Suggested HPLC Gradient Conditions for Electrospray
Suggested
gradient
• For samples not
fractionated
before affinity
column elution
0 to 120 min: 5% to 60% B
120 to 130 min: 60% to 90% B
130 to 131 min: 90% to 5% B
131 to 145 min: 5% B
• For samples
fractionated
before affinity
column elution
0 to 30 min: 5% to 60% B
30 to 35 min: 60% to 90% B
35 to 36 min: 90% to 5% B
36 to 50 min: 5% B
HPLC Gradient Conditions for MALDI Analysis
The following table provides suggested capillary HPLC gradient
conditions for analyzing peptides using a Voyager workstation. Note
that the length of the suggested gradient depends on the complexity of
the sample. Samples that have been further fractionated by cation
exchange can use shorter gradients.
Suggested HPLC Gradient Conditions for MALDIa
QSTAR System Acquisition Method Settings
The following table shows suggested QSTAR System acquisition
method settings for the analysis.
Parameter
Refer to the installation chapter in the InterPlate™
Microfraction Collector for MALDI Analysis User Notes for
details on setting up and connecting the fraction collector.
Suggested Setting
Mobile phase A
0.1% trifluoroacetic acidb in 5% acetonitrile, 95% HPLCgrade water
Mobile phase B
0.1% trifluoroacetic acidb in 95% acetonitrile, 5% HPLCgrade water
Suggested
gradient
Acquisition Method Window (MS Tab)
• For samples
not fractionated
before affinity
column elution
0 to 60 min: 5% to 60% B
60 to 95 min: 60% to 90% B (stop fraction collection)
95 to 96 min: 90% to 5% B
96 to 110 min: 5% B
0 to 30 min: 5% to 60% B
30 to 35 min: 60% to 90% B (stop fraction collection)
35 to 36 min: 90% to 5% B
36 to 50 min: 5% B
Experiment
1
Scan type
TOF MS
Accumulation time
1 second
Experiment
2, 3, and 4
Scan type (for Experiments 2,
3, and 4)
Product Ion
Accumulation Time (for
Experiments 2, 3, and 4)
1 second
a. These gradient conditions are suggested for 1-µL fraction volumes at
1-min intervals. If your fraction collection conditions differ from these,
shorten or lengthen the gradients as required. Do not set fraction-collection
conditions that exceed the MALDI target or InterPlate Microfraction
Collector capacity (maximum of 100 fractions).
CE volts (for Experiments 2,
3, and 4)
45.000 (low end of the mass range)
41.000 (medium part of the mass range)
38.000 (high end of the mass range)
b. If you are performing simultaneous MALDI /electrospray analysis (see
Section 7.6), substitute 0.1% formic acid for the 0.1% trifluoroacetic acid.
• For samples
fractionated
before affinity
column elution
DDE Selection Criteria Dialog Box
Minimum allowed mass
400 amu
Maximum allowed mass
1500 amu
Minimum required intensity
10
Ion(s) to select
Most Intense
Isotope exclusion window
5.0 amu
Mass tolerance window
1.0 amu
Exclusion criteria
Selected
RT exclusion window
2 min
Enable dynamic exclusion
Selected
7.5
Voyager Instrument Method Settings
Use standard reflector-mode instrument settings for your Voyager-DE
PRO, STR, RP, or Elite workstation.
7.6
This section provides guidelines for simultaneous analysis of the ICAT
Reagent-labeled peptides using both a MALDI and an electrospray
system. In this configuration, the capillary reversed-phase HPLC
system is connected to a flow splitter that directs part of the flow to an
InterPlate Microfraction Collector (and on to a Voyager workstation),
with the remaining flow directed to a QSTAR system.
Flow Splitter Output
Set up the flow splitter to deliver a flow rate of 0.5 to 1.0 µL/min
(optimum range for a 300-µm capillary LC column) to the InterPlate
Microfraction Collector. When determining the flow rate, you must
consider the total gradient time and the number of MALDI sample plate
positions to be analyzed.
Analyzing by MALDI
This section provides guidelines for MALDI analysis of the ICAT
Reagent-labeled peptides. In this configuration, the capillary
reversed-phase HPLC system is connected to an InterPlate
Microfraction Collector, then on to a Voyager workstation.
InterPlate Microfraction Collector Guidelines
Refer to the “InterPlate Microfraction Collector Guidelines” in Section
7.5 for details.
InterPlate Microfraction Collector Guidelines
•
•
Analyzing by MALDI and Electrospray
Based on your capillary HPLC column size, set up the
InterPlate Microfraction Collector to collect 0.1- to 2.0-µL
fractions. See “Choosing the Capillary HPLC Column and
Parameters” on page 8.
HPLC Gradient Conditions for MALDI and Electrospray
Analysis
Optimize the HPLC gradient and the fraction collection
according to the total elution time and total collection time for
the peptides of interest. For example, if you collect 100
fractions (the InterPlate Microfraction Collector maximum) at
1 sample per minute, you must select a gradient that elutes the
peptides of interest within a 100-minute period.
Note: For MALDI/electrospray analysis, you must substitute
0.1% formic acid for the 0.1% trifluoroacetic acid in the suggested
gradient.
For gradient-condition details, refer to the “HPLC Gradient Conditions
for MALDI Analysis” table in Section 7.5.
9
Voyager Instrument and QSTAR System Method Settings
Region
Telephone
Fax
Use standard instrument settings for your Voyager workstation. For the
QSTAR System method settings, refer to “QSTAR System Acquisition
Method Settings” in Section 7.4.
Middle Eastern
Countries and
North Africa (Monza,
Italia)
39 (0)39 8389 481
39 (0)39 8389 493
7.7
ICAT Reagent Fragments Using MS/MS
Analysis
Eastern Asia, China, Oceania
MS/MS analysis of ICAT Reagent-labeled peptides results in ICAT
Reagent-specific fragment ions. The structure below shows the typical
fragmentation sites and the corresponding fragment masses of ICAT
Reagents D0 and D8 bound to a cysteine residue in a peptide:
Peptide
Mass + 42
O
HN
328
(332)
NH
NH
S
(D2)
O
CO
O
(D2)
O
284
(288)
227
(227)
O
(D2)
S
NH
(D2)
501
(509)
CH
NH
Cys
403
(411)
61 3 9730 8600
61 3 9730 8799
China (Beijing)
86 10 64106608
86 10 64106617
Hong Kong
852 2756 6928
852 2756 6968
India (New Delhi)
91 11 653 3743/
3744
91 11 653 3138
Korea (Seoul)
82 2 593 6470/6471
82 2 593 6472
Malaysia (Petaling
Jaya)
60 3 758 8268
603 79549043
Singapore
65 896 2168
65 896 2147
Taiwan (Taipei Hsien)
886 2 2358 2838
886 2 2358 2839
Thailand (Bangkok)
66 2 719 6405
66 2 319 9788
477
(485)
Europe
Figure 5 ICAT Reagent-Specific Fragment Ions
8
Australia (Scoresby,
Victoria)
Technical Support
Applied Biosystems is committed to meeting the needs of your
research through enabling technologies such as the ICAT Kit for
Protein Labeling. Our dedicated support staff is available to answer
questions about using this product to its fullest potential.
Contacting Technical Support in North America
By telephone: Dial 1.800.899.5858, press 1, then 3
By FAX: Dial 1.508.383.7855
Austria (Wien)
43 (0)1 867 35 75 00
43 (0)1 867 35 75 11
Belgium
32 (0)2 712 5555
32 (0)2 712 5516
Czech Republic and
Slovakia (Praha)
420 2 61 222 164
420 2 61 222 168
Denmark (Naerum)
45 45 58 60 00
45 45 58 60 01
Finland (Espoo)
358 (0)9 251 24 250
358 (0)9 251 24 243
France (Paris)
33 (0)1 69 59 85 85
33 (0)1 69 59 85 00
Germany (Weiterstadt)
49 (0) 6150 101 0
49 (0) 6150 101 101
Hungary (Budapest)
36 (0)1 270 8398
36 (0)1 270 8288
Italy (Milano)
39 (0)39 83891
39 (0)39 838 9492
Norway (Oslo)
47 23 12 06 05
47 23 12 05 75
Poland, Lithuania,
Latvia, and Estonia
(Warszawa)
48 (22) 866 40 10
48 (22) 866 40 20
Portugal (Lisboa)
351 (0)22 605 33 14
351 (0)22 605 33 15
Contacting the Technical Support Web Site
Russia (Moskva)
7 095 935 8888
7 095 564 8787
1. Access the Applied Biosystems Technical Support web site at
www.appliedbiosystems.com/techsupp.
South East Europe
(Zagreb, Croatia)
385 1 34 91 927
385 1 34 91 840
2. Under the Troubleshooting heading, click Support Request
Forms. Then select the support region for the product area of
interest.
Spain (Tres Cantos)
34 (0)91 806 1210
34 (0)91 806 1206
Sweden (Stockholm)
46 (0)8 619 4400
46 (0)8 619 4401
Switzerland (Rotkreuz)
41 (0)41 799 7777
41 (0)41 790 0676
The Netherlands
(Nieuwerkerk a/d
IJssel)
31 (0)180 331400
31 (0)180 331409
United Kingdom
(Warrington, Cheshire)
44 (0)1925 825650
44 (0)1925 282502
All other countries not
listed (Warrington,
Chesire, UK)
44 (0)1925 282481
44 (0)1925 282509
Japan (Hacchobori,
Chuo-Ku, Tokyo)
81 3 5566 6006
Del.A. Obregon,
Mexico
305-670-4350
3. In the Personal Assistance form, enter the requested information
and your question, then click Ask Us RIGHT NOW.
4. In the Customer Information form, enter the requested information,
then click Ask Us RIGHT NOW.
Contacting Technical Support Outside North America
Region
Telephone
Fax
Africa and the Middle East
Africa (English
Speaking; Fairlands,
South Africa)
27 11 478 0411
27 11 478 0349
Africa (French
Speaking; Courtaboeuf
Cedex, France)
33 1 69 59 85 11
33 1 69 59 85 00
South Africa
(Johannesburg)
27 11 478 0411
Japan
81 3 5566 6505
Latin America
27 11 478 0349
10
305-670-4349
9
Ordering Information
To place an order from the U.S. or Canada, dial 1.800.327-3002, then
follow the voice instructions.
Description
Quantity
Part
Number
3-Assay ICAT Starter Kit for
Protein Labeling (Monoplex
Version)
1 kit
4326692
10-Assay ICAT Kit for Protein
Labeling (Monoplex Version)
1 kit
4326693
ICAT Cartridge Pack–Avidin
5 cartridges
4326694
ICAT Cartridge–Avidin/buffer
pack
1 pack
4326740
ICAT Cartridge Pack–Cation
Exchange
5 cartridges
4326695
ICAT Cartridge–Cation
Exchange/Buffer Pack
1 pack
4326747
Cartridge holder
1 holder
4326688
Needle-port adapter
1 adapter
4326689
Outlet tubing kit
1 kit
4326690
Note: Bulk quantities of ICAT Reagents D0 and D8 are available on
request.
10 References
Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H., Abersold, R.,
1999. “Quantitative Analysis of Complex Protein Mixtures Using
Isotope-Coded Affinity Tags," Nat Biotechnol, 10:994–9.
11
© Copyright 2001, 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.
Applied Biosystems and POROS are registered trademarks, and AB (Design), Applera, QSTAR,
Voyager-DE, Biospectrometry, MicroIonSpray, and InterPlate are trademarks of Applera Corporation
or its subsidiaries in the U.S. and certain other countries.
ICAT is a trademark of the University of Washington and is exclusively licensed to Applied
Biosystems Group of Applera Corporation.
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Printed in the USA, 03/2001
Part Number 4324327 Rev. A