Quantitative proteomics - ICAT

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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

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Master Layout (Part 1)
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This animation consists of 2 parts Part 1
ICAT Part 2 Application of ICAT
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Light (d0) ICAT labeled
Sample 1
Affinity purification
Mixed samples
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Heavy (d8) ICAT labeled
Sample 2
LC-MS/MS analysis
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Affinity purified peptides
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Gygi, S. P. et al., Quantitative analysis of
complex protein mixtures using isotope-coded
affinity tags. Nat Biotech. 1999, 17994-999.
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Definitions of the componentsPart 1- ICAT
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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. 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. 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
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2
Light ICAT reagent
Heavy ICAT reagent
Sample 2
Sample 1
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Action
Audio Narration
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.
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.
As shown in the animation.
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Part 1, Step 2
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Heavy ICAT labeled sample 2
Light ICAT labeled sample 1
Avidin capture agent
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Direction of migration
Affinity purification
Samples mixed
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Affinity purified peptides
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Action
Audio Narration
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.
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.
As shown in the animation.
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Part 1, Step 3
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LC-MS/MS analysis
Column inlet from pump
HPLC Pump
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Injector
Mobile phase
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Sample
Pump
Column
Column elution
LC Column
Sample injector
Column outlet to detector
Sample vials
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Action
Audio Narration
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.
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.
As shown in the animation.
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Part 1, Step 4
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LC-MS/MS analysis
Relative quantification determined by ratio of
peptide pairs.
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Detector
Peptide spectrum
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ESI
Quadrupole (scanning mode)
Collision cell
TOF tube
Reflector
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Action
Audio Narration
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.
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.
As shown in the animation.
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Part 1, Step 5
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MASCOT LC-MS/MS data analysis
proteomics_at_gmail.com
Your name
Email
Proteomics
Search title
Sample protein
Database(s)
SwissProt NCBInr MSDB
Enzyme
Trypsin
Trypsin Chymotrypsin Peptidase
Quantitation
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ICAT
iTRAQ 4plex SILAC ICAT
Taxonomy
Bacterial
Mammalia Bacterial Plant
Fixed modifications
Carboxymethyl (C)
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Variable modification
Oxidation (M)
Da
0.2
C13
Peptide tol.
MS/MS tol.
Da
1.2
Peptide charge
Monoisotopic
Average
Data file
Choose file
Data format
Precursor
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Instrument
Start search
ESI-Q-TOF
MALDI-TOF ESI-Q-TOF MALDI-TOF-TOF

Audio Narration
Description of the action
Action
The MS/MS data analysis shareware has some extra
inputs such as 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.
As shown in animaion.
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.
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Part 1, Step 5
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ADVANTAGES
LIMITATIONS
Relative protein levels between samples estimated
within 10 accuracy
Bias for Cys-rice proteins
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Large ICAT reagent interferes with MS
fragmentation
Useful for complex protein mixtures
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Highly automated
Tag size reduced quality of MS data
Peptides sequenced directly using MS/MS
Expensive and non-specific
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Action
Audio Narration
Description of the action
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.
The text boxes must appear one at a time under
their respective column headings as shown in
animation.
As shown in the animation.
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Master Layout (Part 2)
This animation consists of 2 parts Part 1
ICAT Part 2 Application of ICAT
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Plasma sample of normal, healthy control
Immunodepleted plasma
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Immune-affinity column chromatograpy
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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
Plasma sample from breast cancer patient
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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, 10114.
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Definitions of the componentsPart 2-
Application of ICAT
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• 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
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Immune-affinity column chromatograpy
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Plasma sample of normal, healthy control
Plasma sample from breast cancer patient
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Immunodepleted plasma
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Action
Audio Narration
Description of the action
The authors obtained plasma proteomes from 6
breast cancer patients and 6 healthy controls.
These plasma samples were first treated on an
immune-affinity column in order to deplete them
of their high abundance proteins. The
immunodepleted serum samples were then used for
further analysis by ICAT.
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.
As shown in the animation.
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Part 2, Step 2
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Plasma sample from breast cancer patient
Plasma sample of normal, healthy control
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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
Audio Narration
Description of the action
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.
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.
As shown in the animation.
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Interactivity option 1Step No 1
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Drag drop the components required for ICAT
labeling and analysis in their correct order in
the boxes given below.
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Results
Boundary/limits
Interacativity Type Options
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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User has to drag and drop the images shown above
into the correct boxes below.
Drag drop.
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Questionnaire
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• 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?
• 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
• 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
• 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 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

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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, 17994-999.
• 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, 10114.
• 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,
  2796-804.
• 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, 622451-70.