Transcript appendix 1

Methodology for Isobaric tag for relative and
absolute quantitation (iTRAQ)
The identification and quantitation of complex protein mixtures have
been facilitated by MS-based quantitative proteomic techniques.
Isobaric tag for relative and absolute quantification (iTRAQ) consists
of amine-specific, stable isotope reagents that can label peptides of
upto four to eight different biological samples.
 Related Los: Isobaric tag properties, Trypsin properties
> Prior Viewing – IDD-6. Extraction of serum protein, IDD-11. Protein quantification,
IDD-28. In solution digestion, IDD-34. Liquid chromatography - Ion exchange
Future Viewing – IDD-31. MALDI-TOF data analysis, IDD-39. LC-MSMS data
analysis
>
 Course Name: Isobaric tag for relative and absolute quantitation (iTRAQ)
 Level(UG/PG): PG
 Author(s): Dinesh Raghu, Vinayak Pachapur
 Mentor: Dr. Sanjeeva Srivastava
*The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license
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Definitions and Keywords
1. Isobaric Tag for Relative & Absolute Quantification: iTRAQ is a MS based technique
for relative and absolute quantification of proteins present in up to four cell preparations by
making use of four isobaric, isotope-coded tags that label the proteins via their N-terminal.
2. Protein samples: The samples whose proteins need to
procedure.
be quantified by iTRAQ
3. Trypsin digestion: Trypsin is a proteolytic enzyme that cleaves proteins at the C-terminal
of arginine and lysine residues except when they are followed by proline. This enables large
protein samples to be broken down in to small peptide fragments.
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4. Peptide fragments: The smaller fragments obtained upon cleavage of the protein
samples after trypsin digestion.
5. iTRAQ label: The iTRAQ reagent consists of a protein reactive group that labels the Nterminus of all peptides as well as free amine groups of lysine side chains, a neutral balance
portion and a reporter group, giving it a total mass of 145. The different distribution of
isotopes between the reporter and balance groups makes the labels isobaric and enables
their detection upon fragmentation and release in MS.
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Definitions and Keywords
6. SCX purification: Tagged peptides are fractionated on a strong cation exchange column to
remove any unbound iTRAQ reagent and to simplify the peptide mixture.
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7. LC-MS/MS analysis: The iTRAQ labeled peptides obtained are further purified by reverse
phase liquid chromatography and then analyzed by tandem MS. Each tag releases a distinct
mass reporter ion upon peptide fragmentation, the ratio of which determines the relative
abundances of the peptides.
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Learning objectives
After interacting with this learning object, the learner will
be able to:
1.
Define sample processing using trypsin digestion
2.
Carry out sample labelling with multiplex reagents
3.
Perform sample run through chromatography
4.
Infer data analysis through MALDI instrument
5.
Assess the troubleshooting steps involved in the
experiments.
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Master Layout
Slide
6-7
Sample processing
Slide
8-15
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Sample labelling
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4
Slide
16-18
Cat-ion exchange chromatography
Slide
19-20
Liquid chromatography
Slide
21-23
MALDI-MS
Slide 24
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Data analysis and interpretation
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Step 1:
T1:Sample processing
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Sample: A
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B
Description of the action/ interactivity
Take user through the slides of IDD-6.
Extraction of serum protein, IDD-1.
Extraction of bacterial protein and
IDD-11. Protein quantification. Now
display 4 tubes showing 2-3ug/ml of
protein concentration.
C
D
Audio Narration
(if any)
At max 4/8 different protein
samples can be run together or
analysed under the iTRAQ.
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Step 1:
T1:Sample processing
C-terminal
N-terminal
Trypsin
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Protein
Trypsin
enzyme
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Description of the action/ interactivity
Take user through the slides of IDD-28. In
solution digestion. Animate the tubes kept at
37’C overnight. First show the four tubes with
different colored ‘protein samples’ in them. let
user adds trypsin into each tubes. The
mechanism of trypsin action must be zoomed.
Animate the orange ‘trypsin’ moving across the
red band from N-terminal to C-terminal. And
makes a cut along the green region to give out
smaller fragments/peptides. Similarly animate
for other samples with user controller.
Digested
fragments
Audio Narration
(if any)
During In-solution digestion, trypsin
is acted upon the sample, which
does the protein digestion at
specific sites. long chain proteins
are broken down to small chain
peptides.
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Step 2:
Ethanol
bottle
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T2:Sample labelling
TEAB
bottle
balance
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Description of the action/ interactivity
Instruct user to prepare labeling buffer. Let user
take out TEAB bottle, ethanol bottle and 100ml
measuring cylinder from the rack to place it near
the balance. Now let user weigh 22.77mg of
TEAB with help of spatula, transfer the weighed
amount to tube. Now let user measure 750ul of
ethanol with pipette and add to the tube, now
add 250ul of water to make the volume to 1ml in
the tube and mark it has labeling buffer.
labeling buffer
Audio Narration
(if any)
Labelling buffer is used for the
labelling reaction, which helps to
provide the buffer conditions.
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Step 2:
T2:Sample labelling
Isotopes
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methylpiperazine
acetic acid
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balance
Description of the action/ interactivity
Instruct user to prepare Multiplex reagent. Let
user take out methylpiperazine acetic acid and
isotopes from the rack and place it near the
balance. Now let user weigh 1mg of isotopes
and add into a tube containing 1ml of
methylpiperazine acetic acid. Vortex the tube for
a brief spin and label it has multiplex reagent.
Repeat the same protocol for all other isotopes
to be used in the experiments.
multiplex reagent
Audio Narration
(if any)
The concentration of multiplex
reagent depends on the choice of
user for each of theexperiment. The
final % of Weight/volume can be
altered and standardized.
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Step 2:
T2:Sample labelling
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labeling buffer
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multiplex reagent
Sample: A
Description of the action/ interactivity
Instruct user to label the samples. Let user take
around 150ug of each sample protein into a
fresh tube and label it accordingly, now resuspend it in 100ul of Labeling buffer with the
help of pipette. To this solution add 1ml of
multiplex reagent. Let user mix and vortex each
of the tube and keep the tube at room
temperature for 30min.
B
C
D
Audio Narration
(if any)
Each sample must be labeled with
different isotopes and kept at room
temperature for labeling reaction to
happen.
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Step 2:
T2:Sample labelling
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Description of the action/ interactivity
Show the smaller colored fragments in the
beakers above. The chemical structure shown
must be added to each of the beakers to
generate the fragments shown in the beakers
below.
Audio Narration
(if any)
The mechanism of binding is explained in
next slide.
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Step 2:
T2:Sample labelling
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Description of the action/ interactivity
Display the above figures, In “A” for iTRAQ
reagent. Animate the “reporter group mass”,
“Balance Group” combined of both these
group is “Isobaric tag total mass” (114117+31-48) along with “Amine specific
peptide reactive group” followed with audio
narration in black color text. In fig “B”
display the reporter group mass kept
constant with isotopic elements followed by
audio narration in green color text. In fig “C”
show the formation of isotopes ready to
react with peptide followed by audio
narration in red color text.
Audio Narration
(if any)
The iTRAQ reagent consists of a reporter group, a balance
portion and a peptide reactive group that interacts with the
N-terminus of the peptide or free amino group of Lysine
residues, giving it an overall mass of 145. The overall mass
of reporter and balance is kept constant by using
differentially isotopic enrichment with C, N and O. thereby
enabling the labeling and quantification of four samples
simultaneously. reporter group ranges in mass/charge from
114.1 to 117.1, while the balance group ranges in mass
from 28 to 31 Da, with over all constant combined mass
(145.1 Da) for each of the four reagents. This has been
further improved to allow labelling of eight samples
simultaneously.
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Step 2:
T2:Sample labelling
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Description of the action/ interactivity
Display the above figures, the peptide of each
sample going and replacing the NHS ester,
final image display the peptide bound to the
reporter and balance group.
Audio Narration
(if any)
The tag reacts with a peptide forms
an amide linkage with any peptide
amine. The peptide amine may be a
N-terminal or amino group of lysine.
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Step 2:
T2:Sample labelling
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distilled water
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Description of the action/ interactivity
After 30min of labelling reaction, Instruct user
to carry out Quenching step. Let user takes out
distilled water, set the pipette to 350ul and add
set volume to each reaction unit. Now animate
the free tags binding to water molecules.
Audio Narration
(if any)
Quench reaction is used to stop
excess labelling. By using water the
excess labelling of peptides is
stopped.
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Step 2:
T2:Sample labelling
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Description of the action/ interactivity
Instruct user to “pool/mix the samples”. Let
user take out full volume from the each
reaction unit with pipette and transfer into fresh
common tube, let user label it has pooled
samples.
Audio Narration
(if any)
After the quenching the reaction, the
samples need to be pooled before
running into the instrument.
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Step 3:
T3:Cat-ion exchange chromatography
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Description of the action/ interactivity
Instruct user to carry out Cation
chromatography, for more information let user
follow the IDD-34. Liquid chromatography - Ion
exchange. Instruct user to transfer the pooled
sample into the exchange column. Animate the
combined/pooled sample passing through the
cat-ion exchange column, let tags with circles
colored in red get holdup into the column and
the tag attached with peptide pass out of the
column. Let user collect the sample.
Audio Narration
(if any)
The pooled sample is taken to cat-ion
exchange column for purification. The
excess and unbound tags need to be
removed from the analysis, for this
reason stationary phase specific to
tags is used for the separation. After
the sample collection SDS-PAGE run
is carried out to separate out
proteins.
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Step 3:
T3:Cat-ion exchange chromatography
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Description of the action/ interactivity
Setup: strong cation exchange, polysulfoethyl
A column (4.6X100mm, 5um, 300 Åmstrong).
Let user dissolve the sample in 4ml loading
buffer. Linear gradient of 0-500mm KCL (25%
v/v CAN, 10mM kh2PO4) over 15min at flow
rate of 1ml/min. let user collect the fraction at
1min interval
Audio Narration
(if any)
Loading buffer composition (25% v/v
ACN, 10 mM KH2PO4, pH 3, with
phosphoric acid) depends on the
sample complexity.
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Step 3:
T3:Protein separation
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Description of the action/ interactivity
After getting sufficient amount of protein
concentration, Instruct user to carry out
SDS-PAGE run for the sample from day 1.
Take user through the IDD:18 SDS-PAGE
for protein separation. Animate the SDSPAGE gel with blue bands at different
regions along the gel. Let user do the
manual picking please refer IDD: 26 Spot
picking and carry out trypsin digestion like
in IDD:27 In-gel digestion.
Audio Narration
(if any)
SDS-PAGE sample loading buffer:
0.3 M Tris–HCl, pH 6.8,
10% SDS, 12.5% vol/vol 2mercaptoethanol, 50% glycerol,
0.016% bromophenol blue Depending
upon user set up, one can carry to
resolve protein sample through SDSPAGE followed by in-gel trypsin
digestion to carry out MS or directly
can do In-solution trypsin digestion
followed by MS analysis.
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Step 4:
T4:Liquid chromatography
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Description of the action/ interactivity
Display the figure above of HPLC unit, for more
information user can follow IDD:41 Liquid
chromatography. Animate user injecting the
sample into he instrument, now along with
mobile phase sample must move into the
column, in the column show the separation of
peptides from one another, like one peptide
eluting out faster and other slower, the peptide
must be collected into the sample vials.
Audio Narration
(if any)
The sample peptides are eluted out
or separated out from one other
depending on the binding properties
of peptide to the stationery phase.
The retention and elution properties
of the peptide help in the separation
by controlling the flow rate of mobile
phase with the help of pump. The
separated peptide are collected into
each sample vials separately.noe the
samples are ready for the MALDI-MS
analysis.
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Step 4:
T4:Liquid chromatography
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Description of the action/ interactivity
LC set up: MALDI spotting device, trap column
0.3X5mm with C18 packing, eluted onto
0.1X150mm analytical column with C18
packing. Automated binary gradient(800nl/min)
from 95% buffer A(2%Acetonitrile, 0.1% TFA)
to 45% buffer B(85%Acetonitrile, 0.1% TFA,
5% isopropanal) over 35min
Audio Narration
(if any)
LC setup depends on the sample
type and varies accordingly with the
experiments.
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Step 5:
T5:MALDI-MS
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Description of the action/ interactivity
The sample need to be mixed with matrix
before analysis. For sample preparation please
go through IDD-29. Matrix preparation for
MALDI analysis. Once the sample preparation
is done it need to be loaded on to MALDI plate
for the firing of spots. Sample preparation with
1:2 ratio.
Audio Narration
(if any)
Before the MALDI analysis the
sample peptide need to be mixed
with matrix to make up the solution
which is compatible for the MALDI
analysis. Once the solution is
prepared the firing of spot need to be
carried out.
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Step 5:
T5:MALDI-MS
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Description of the action/ interactivity
For firing of sample spots and data acquisition
please go through IDD: 60 MALDI
instrumentation.
Audio Narration
(if any)
Firing of spots are carried out to
collect the spectrum of peptides.
Once firing of sample peptide takes
place, the peptides are separated
and detected depending on their
mass and time taken to travel the
path before getting detected. In the
detector the data is generated giving
information of the mass/charge of
each single peptide with its intensity.
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Step 5:
T5:MALDI-MS
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Description of the action/ interactivity
For firing of sample spots and data acquisition
please go through IDD:30 MALDI
instrumentation and IDD:39 LC-MS/MS data
analysis. For the image above animate, firing
of isotope bind to peptide being fired and
detected as identical peak in MS. Later, after
MS/MS. For MS/MS: collision energy of 1kV,
collision gas pressure of 1.5X10-6 Torr.
Audio Narration
(if any)
In initial MS user acquires a single,
unresolved peak for mixture of four
identical peptides. Followed by
MS/MS, the four reporter group ions
appear as separate peaks. The
reporter ions intensities thus deduces
the relative concentration of the
peptides.
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Step 6:
T6:Data analysis and interpretation
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Description of the action/ interactivity
For data analysis and interpretation please go
through IDD:31 MALDI data analysis.
For fixed modification: let user select Sacetamido, N-terminal, and lysine. In case of
Variable modification let user select:
methionine oxidation, with one missed
cleavage and precursor error tolerance around
50 ppm.
Audio Narration
(if any)
After collection of spectrum, protein
need to be identified with help of
database search. Once protein
identification is done, user can
correlate the proteins for the study.
Slide
1-5
Introduction
Slide
8-15
Slide
6-7
Tab 01
Tab 02
Slide
16-18
Tab 03
Slide
19-20
Slide 24
Slide
21-23
Tab 04
Tab 05
Tab 06
Name of the section/stage
Interactivity
area
Animation area
I1 slide-18: In case of complex sample ask for the suggestions from the user for
further steps?
Instruction: a) in solution isoelectric focusing
Button 01
Button 02
b) Desalt the sample.
c) increase Acetonitrile concentration during sample loading into
the column.
Button 03
d) reverse phase HPLC.
are the best option in case of complex sample.
I2 slide-23: In case of fragment harder and more stable, ask user input?
Instruction: for any given labeled peptides mass, it is better to increase
collision energy slightly to give equivalent fragmentation and increase the
intensity of reporter ions is te best solution.
Instructions/ Working area
Credits
APPENDIX 1
Questionnaire:
1. Which type of chromatography is used for removal of excess iTRAQ reagent prior to
LC-MS/MS?
Answers: a) Reverse phase
b) Gel filtration
c) Strong cation exchange
d) Affinity chromatography
2. The total mass of the iTRAQ reagent is:
Answers: a) 140
b) 143
c) 148
d) 145
APPENDIX 1
Questionnaire:
3. Which region of the iTRAQ reagent has variable mass value?
Answers:
a) Reporter group
b) Peptide-reactive group
c) Balance region
d) None of the above
4. In iTRAQ, Quantitation is performed at which stage?
a) MS stage
b) MS/MS stage
c) TOF stage
d) Firing stage
APPENDIX 1
Questionnaire:
5. Which of the following statements regarding iTRAQ analysis is incorrect?
Answers: a) iTRAQ can label all peptides in up to ten different biological samples
b) iTRAQ reaction labels the N-terminus of peptides
c) There is no reduction of peptides based on amino acid composition
d) Reporter group is lost during fragmentation
6. Better method for Quantitation of peptide is:
Answers: a) MALDI
b) LC
c) iTRAQ
d) ICAT
APPENDIX 2
Research papers:
•Lynn R. Zieske, A perspective on the use of iTRAQ TM reagent technology for protein complex
and profiling studies, Jour Exp Botany, 2006, 57:1501–1508.
•Philip L. Ross et. al., Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using
Amine-reactive Isobaric Tagging Reagents, Mol & Cell Proteomics 3.12, 1154-1169.
•Wiese, S. et al., Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in
proteome research. Proteomics 2007, 7: 340-350.
•Boylan, K. L. M. et al., Quantitative proteomic analysis by iTRAQ for the identification of
candidate biomarkers in ovarian cancer serum. Proteome Science 2010, 8:31.
•http://www.matrixscience.com/pdf/2010WKSHP2.pdf
APPENDIX 3
Summary
Use of four different isobaric peptide derivatization reagents for a complex protein
mixture, yields a very informative MS data for protein identification and
quantification. The nature of isobaric tags provide a simple comparison of
multiple same (probably sample), without any increase in complexity of
chromatography or MALDI-MS.