Transcript Part 1

Quantitative proteomics ICAT
a
The identification and quantitation of complex protein
mixtures have been facilitated by MS-based quantitative
proteomic techniques. Isotope coded affinity tag consists
of chemical labeling reagents that specifically label a
defined amino acid side chain, a linker and a tag group.
Harini Chandra
Master Layout (Part 1)
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2
This animation consists of 2 parts:
Part 1: ICAT
Part 2: Application of ICAT
Light (d0)
ICAT labeled
Sample 1
Mixed samples
Affinity
purification
3
Heavy (d8)
ICAT labeled
4
5
Relative abundance
Sample 2
LC-MS/MS analysis
Affinity purified
peptides
Retention time
Gygi, S. P. et al., Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotech. 1999,
17:994-999.
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Definitions of the components:
Part 1- ICAT
1. Isotope Coded Affinity Tagging (ICAT): ICAT is an in vitro labeling technique that modifies
peptides or proteins specifically at the cysteine amino acid residue and can be used for
accurate quantitation of protein expression.
2. Samples 1 & 2: The protein samples that need to be tagged and studied.
3. Light ICAT label: The light ICAT reagent consists of a Cys-reactive group, an ICAT linker
consisting of hydrogen atoms and a biotin tag. The chemically reactive group forms covalent
bonds with peptides or proteins while the affinity tag enables the protein to be isolated by
affinity chromatography in a single step.
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4. Heavy ICAT label: The heavy ICAT label consists of a Cys-reactive group, an ICAT linker
consisting of heavy deuterium isotope and a biotin tag.
5. Affinity purification: A chromatographic purification procedure that makes use of specific
interactions between the analyte of interest and the capture reagent immobilized on the
column. In ICAT, avidin affinity chromatography is employed due to its specificity of interaction
with biotin.
6. Affinity purified peptides: The peptides obtained after purification on the affinity column.
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7. LC-MS/MS analysis: The peptides obtained are further purified by liquid chromatography
and then analyzed by tanderm MS. Both quantity and sequence identity of proteins from which
the tagged peptides were obtained can be determined.
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Part 1, Step 1:
2
Light ICAT
reagent
Heavy ICAT
reagent
Sample 2
Sample 1
3
O
Light linker
chain
Biotin tag
HN
NH
O
H
S
4
Action
5
As
shown in
the
animatio
n.
N
H
H
O
HN
NH
H
H
OO
H
O
Sulfhydryl
reactive group
Heavy
Sulfhydryl
Biotin
tag
reactive group linker chain
O
S
O H
Description of the action
First show the two beakers with the green and
purple circles in them. Next show the hand on
top which must add drops to the sample and
the three black lines must appear which must
be zoomed into to show the figure below. This
must be repeated for Sample 2 as well as
shown in animation.
H
H
N
NH
H
D
D
D
D
O
D
O
O
O D
D
D
N
H
Audio Narration
ICAT is an in vitro labeling procedure that involves
tagging of protein or peptide samples with the
ICAT reagent specifically at their Cys residues.
The ICAT reagent consists of a biotin tag, a light or
heavy linker chain and a Cys-reactive group. One
sample is tagged with the light ICAT reagent while
the other is tagged with heavy ICAT.
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Part 1, Step 2:
Light ICAT
labeled sample 1
Heavy ICAT
labeled sample 2
Avidin capture
agent
2
Samples mixed
Affinity
purification
Direction of
migration
3
Affinity purified
peptides
4
Action
5
As
shown in
the
animatio
n.
Description of the action
Show the contents of the two beakers on
top being mixed into the empty beaker
shown below. This must then be poured
into the column shown on the right. The
contents must flow through the column
into the empty beaker below. The samples
must be modified as shown in diagram.
Audio Narration
The labeled samples are mixed together and then
cleaved enzymatically to generate peptide
fragments, some of which will contain the ICAT
tag. These are purified by means of affinity
chromatography, which makes use of the specific
interaction between immobilized avidin on the
column and biotin of the linker.
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Part 1, Step 3:
LC-MS/MS analysis
Column inlet
from pump
HPLC Pump
2
Injector
Mobile phase
3
Sample
LC Column
Sample injector
elution
Sample vials
4
Action
5
Pump Column
As
shown in
the
animatio
n.
Description of the action
Show the beaker on top followed by the arrow. Then show the setup
below with all its labels. The second and third boxes must be
zoomed into to show the figures on the right. The ‘injector’ must
enter the sample bottle with its plunger down. It must remain in this
bottle for a couple of seconds and the plunger must be shown to
move up. This must then move and be injected into the column.
Liquid must be shown to flow through the tube connecting the ‘pump’
and ‘column’. Once the liquid flows, the colour in the column must
change and the liquid must be shown to pass through the tubing at
the outlet.
Column
Column outlet to
detector
Audio Narration
Further purification of the affinity purified
peptides
is
carried
out
by
liquid
chromatography wherein the sample is passed
through a column containing a packed
stationary phase matrix that selectively adsorbs
only certain analyte molecules. The eluted
fractions are further characterized by MS.
LC-MS/MS analysis
Relative quantification
determined by ratio of
peptide pairs.
Relative abundance
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Part 1, Step 4:
2
Detector
Retention time
Peptide spectrum
3
m2
ESI
m3
Quadrupole
(scanning
mode)
4
Action
5
m4
m1
As
shown in
the
animatio
n.
Collision cell
TOF tube
Description of the action
First show all the components of the instrument – the syringe,
four rods, cube, blue rectangle, gray square with the dotted lines
& the detector. Next show appearance of the coloured circles.
Only the red one must move through the rods and after entering
the rectangular box, it must be fragmented to give smaller circles.
These must migrate through the blue tube and get reflected to
reach the ‘detector’. The smallest circles must move the fastest
while the largest must move slowest. Once it reaches the
detector, the graph on top must be shown.
Reflector
Audio Narration
The purified peptide fragments are
then analyzed by MS/MS. Both the
quantity and sequence identity of the
of the proteins from which the tagged
peptides were obtained can be
determined. This technique can be
used for complex protein mixtures.
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Part 1, Step 5
MASCOT LC-MS/MS
data analysis
Search title
Sample protein
Enzyme Trypsin
Trypsin
Quantitation ICAT
Chymotrypsin
iTRAQ 4plex
Peptidase
Taxonomy Bacterial
SILAC
Mammalia
ICAT
Carboxymethyl (C)
Fixed
Bacterial
modifications
Plant
Database(s) SwissProt
NCBInr
MSDB
2
Variable
modification
3
Oxidation (M)
Peptide tol.
1.2
Data file
Data format
Instrument
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Action Description of the
As shown
in
animaion.
Da
# C13
MS/MS tol. 0.2
Monoisotopic
Peptide charge
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Email [email protected]
Your name Proteomics
Da
Average
Choose file
ESI-Q-TOF
Precursor
Start search…
MALDI-TOF
ESI-Q-TOF
action
Audio Narration
MALDI-TOF-TOF
The MS/MS data analysis shareware has some extra inputs such as
First show the computer with the
screen having a form on the inside.
This must be zoomed into and the
form above must be displayed. Each of
the fields must be filled in as shown
with some requiring selection using the
white mouse pointer as depicted.
Quantitation, MS/MS tolerance, peptide charge, instrument etc. in addition to
the fields for PMF. They require inputs from the user regarding the
experimental parameters used such as enzyme cleavage, protein name,
modifications etc. and the desired search criteria like taxonomy, peptide
tolerance etc. Commonly used protein databases against which the MS
information is processed to retrieve sequence data include NCBI, MSDB and
SwissProt. The data file generated from MS is uploaded and the search
carried out.
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Part 1, Step 5:
ADVANTAGES
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Relative protein levels between
samples estimated within 10%
accuracy
Useful for complex protein mixtures
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LIMITATIONS
Bias for Cys-rice proteins
Large ICAT reagent interferes with
MS fragmentation
Highly automated
Tag size reduced quality of MS
data
Peptides sequenced directly using
MS/MS
Expensive and non-specific
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Action Description of the action
5
As
shown in
the
animatio
n.
The text boxes must appear
one at a time under their
respective column headings as
shown in animation.
Audio Narration
ICAT is an accurate technique that can be useful for determining
complex protein mixtures. It is highly automated allowing peptides to
be sequenced directly using tandem MS. However, the major limitation
for ICAT is its bias for cysteine rich proteins. The large ICAT reagent
often interferes with MS fragmentation and reduces the quality of the
MS spectra obtained. This limitation has, however, been successfully
overcome through development of a cleavable ICAT (cICAT) reagent
which is removed prior to MS fragmentation and analysis.
Master Layout (Part 2)
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This animation consists of 2 parts:
Part 1: ICAT
Part 2: Application of ICAT
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Plasma sample
of normal,
healthy control
3
ICAT
Immune-affinity
column
chromatograpy
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Plasma sample
from breast
cancer patient
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Immunodepleted
plasma
155 proteins identified of
which, 33 showed 1.5-fold
abundance changes in plasma
of breast cancer patients
compared to healthy controls
Kang, U. B. et al., Differential profiling of breast cancer plasma proteome by isotope-coded affinity tagging method reveals
biotinidase as a breast cancer biomarker. BMC Cancer 2010, 10:114.
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Definitions of the components:
Part 2- Application of ICAT
1. Normal healthy control: Normal healthy control refers to those who do not have the
disease/condition that is being studied. The authors made use of plasma proteomes obtained
from 6 healthy control samples.
2. Breast cancer patients: Plasma samples from 6 patients with breast cancer were analyzed
using ICAT labeling technique.
3. Immune-affinity column chromatography: Immune-affinity column chromatography is a
process that is carried out in order to remove the high abundance proteins present in sera,
which tend to hamper the process of detection of medium or low abundance protein markers.
Antibodies specific to the high abundance proteins of interest are immobilized on the column
and used to specifically remove them.
4. Immunodepleted serum: The serum from which the high abundance proteins have been
removed, leaving behind only the medium and low abundance proteins thereby reducing the
dynamic range, is known as immunodepleted serum.
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Part 2, Step 1:
Immune-affinity
column
chromatograpy
2
Plasma sample
of normal,
healthy control
Plasma sample
from breast
cancer patient
3
Immunodepleted
plasma
4
Action
5
As
shown in
the
animatio
n.
Description of the action
First show the tubes on top with their components
followed by the column in the centre. These
components must be passed through the column, one
tube at a time. For the left tube only the brown and pink
circles must come out as shown while the remaining
must stay in the column. For the tube on the right, the
green circles must come out in addition to the brown
and pink while rest must remain in the column.
Audio Narration
The authors obtained plasma proteomes from 6
breast cancer patients and 6 healthy controls. These
plasma samples were first treated on an immuneaffinity column in order to deplete them of their high
abundance proteins. The immunodepleted serum
samples were then used for further analysis by
ICAT.
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Part 2, Step 2:
ICAT
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155 proteins identified of which, 33 showed 1.5-fold
abundance changes in plasma of breast cancer
patients compared to healthy controls.
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Action Description of the action
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Plasma sample from
breast cancer patient
Plasma sample of
normal, healthy control
As
shown in
the
animatio
n.
First show the tubes on top followed by the
arrows towards the blue hexagon “ICAT”.
This must be followed by appearance of
the red text box below.
Audio Narration
The immunodepleted plasma proteome samples were used for
analysis by ICAT. 155 proteins were identified of which 33
showed 1.5-fold abundance changes in plasma of breast cancer
patients as compared with the healthy controls. Biotinidase was
found to be significantly down regulated in breast cancer
patients.
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Interactivity option 1:Step No: 1
Relative abundance
Drag & drop the components required for ICAT labeling and analysis in their
correct order in the boxes given below.
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RP-HPLC
3
O
HN
Retention time
MS/MS analysis
NH
S
ICAT label
Purification
column
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2
3
Options
Boundary/limits
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4
Interacativity Type
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Drag & drop.
User has to drag and
drop the images shown
above into the correct
boxes below.
Results
User has to drag & drop the images above
into the correctly numbered boxes below.
Correct answer is as displayed in the
animation. If the user drags the wrong image
into the box, a red cross must appear over it
and it must return back to its original position.
Every time the user drags a correct image into
the box, a green tick must appear over it.
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Questionnaire
1. Which of the following amino acid residues is modified during ICAT analysis?
Answers: a) Aspartic acid b) Lysine c) Cysteine d) Proline
2. Which chromatographic technique is employed to purify the labeled peptide fragments?
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Answers: a) Strong cation exchange b) Gel filtration c) Hydrophobic interaction chromatography
d) Affinity chromatography
3. One of the major drawbacks of the ICAT technique is
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Answers: a) Bias for Cys-rich proteins
b) Cannot be used for complex mixtures
c) Extremely cumbersome due to many manual steps
d) Requirement for multiple purification steps
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4. In the study by Kang et al., the levels of biotinidase was found to be:
Answers: a) Up regulated b) Down regulated c) Not affected d) None of the above
5. The cleavable ICAT reagent was developed to overcome which of the following drawbacks?
Answers:
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a) Bias of regular ICAT reagent for Cys-rich proteins
b) Normal reagent cannot be used for complex mixtures
c) Interference of the ICAT reagent with MS spectra due to large size
d) Requirement for multiple purification steps using the normal reagent.
Links for further reading
Research papers:
•
Gygi, S. P. et al., Quantitative analysis of complex protein mixtures using isotope-coded
affinity tags. Nat Biotech. 1999, 17:994-999.
•
Kang, U. B. et al., Differential profiling of breast cancer plasma proteome by isotope-coded
affinity tagging method reveals biotinidase as a breast cancer biomarker. BMC Cancer
2010, 10:114.
•
Rivera-Monroy, Z. et al., Fluorescent isotope-coded affinity tag (FCAT). I: Design and
synthesis. Bioorg. Chem. 2008, 36(6):299-311.
•
Gasparri C. et al., Proteomics reveals high levels of vitamin D binding protein in myocardial
infarction. Front Biosci (Elite Ed). 2010, 2:796-804.
•
Butler, G. S. et al., Identification of cellular MMP substrates using quantitative proteomics:
isotope-coded affinity tags (ICAT) and isobaric tags for relative and absolute quantification
(iTRAQ). Methods Mol. Biol. 2010, 622:451-70.