iTRAQ is a quantitative proteomics technique that uses isobaric tags to label peptides from up to 8 samples for identification and quantification by mass spectrometry. The iTRAQ reagent contains four amine-reactive labels with different reporter masses but the same overall tag mass. Labeled peptides are indistinguishable by mass but produce distinct reporter ions upon fragmentation, allowing relative quantification of proteins across samples. The technique provides advantages over previous methods through multiplexing, precision, and expanded proteome coverage, though errors can arise from variability in sample processing and digestion efficiency.

1. ISOBARIC TAG FOR RELATIVE AND
ABSOLUTE QUANTIFICATION (iTRAQ)
ASHOKKUMAR P
I – MSc Genetics & Plant Breeding
2022508002

2. iTRAQ
• Isobaric Tag for Relative and Absolute
quantification
• The identification and quantification of complex
protein mixtures have been facilitated by the mass
spectrometry based quantitative proteomic
techniques.
• The iTRAQ reagent consists of amine specific stable
isotope reagents that can label peptides of upto 4
to 8 different biological samples.
Isobaric tags for relative and absolute quantitation
(iTRAQ) is an isobaric labeling method used in
quantitative proteomics by tandem mass
spectrometry to determine the amount of
proteins from different sources in a single
experiment. It uses stable isotope labeled
molecules that can be covalent bonded to the N-
terminus and side chain amines of proteins.

3. iTRAQ
• Isobaric Tag for Relative and Absolute
quantification
• The identification and quantification of complex
protein mixtures have been facilitated by the mass
spectrometry based quantitative proteomic
techniques.
• The iTRAQ reagent consists of amine specific stable
isotope reagents that can label peptides of upto 4
to 8 different biological samples.

4. iTRAQ
• Isobaric Tag for Relative and Absolute
quantification
• The identification and quantification of complex
protein mixtures have been facilitated by the mass
spectrometry based quantitative proteomic
techniques.
• The iTRAQ reagent consists of amine specific stable
isotope reagents that can label peptides of upto 4
to 8 different biological samples.

5. Quantitative Proteomics : Invitro
labelling methods
• Invitro labelling method rely on use of labelling reactions at a specific site in
proteins or peptides, based on various labellling chemistry different types of
strategies have been developed to introduce isotopes at either protein or
peptide level.

6. Use of Mass Spectrometry in iTRAQ
• Mass Spectrometry has played avery major role in
proteomics and now it is becoming a very essential tool
to study the complex biological system in various
diseases.
• iTRAQ is a mass spec based technique for relative and
absolute quantitation of proteins present in upto 4 to 8
samples depending upon the type of iTRAQ tags, and
these labels can be provided in the proteins where the
N-terminal.
• The iTRAQ and ICAT are the only currently available
commercial tagging technologies, where quantitation
can be carried out in the MS/MS mode.

7. iTRAQ
• The iTRAQ technique was first time described by
Ross et. al, in 2004 and it was subsequently
commercialized by the applied biosystems.
• These iTRAQ reagents are set of multiplexed,
amine-specific isotope reagents.
• It enables simultaneous identification and
quantitation, both relative and absolute.
• There are two types of iTRAQ reagents –
• 4-plex for processing up to 4 samples
• 8-plex for processing up to 8 samples

8. iTRAQ method
• In iTRAQ all derivativatized peptides of a given
sequence are isobaric and coeluted (derived from
control and treatment biological samples)
• Upon collision induced dissociation in MS/MS
experiments, it provides reporter ions(signature)
that differ in m/z value( reporter ions can be used
to monitor the relative quantitation for proteins)

9. iTRAQ Reagent
• There are a set of four isobaric amine specific labelling reagents 114,115,116 and 117.
• The reagent consists of a reporter group, a balancer group, and a peptide reactive group.
• The protein group labels the n terminals of all peptides as well as the free amine group of lysine side chains.
• The neutral balance portion and the reporter group provide total mass of 145, so this method can allow multiplexing of upto 4 or 8
different samples in a single LC MS/MS experiment.
• The different distribution of isotopes between the reporter and balance group makes the label isobaric and it enables the detection
upon fragmentation and the release in mass spec.

10. Reporter
Provides signature ion in MS/MS
Maintains the charge state and ionization efficiency of peptide
Balancer
Balances mass charge of reporter to provide total mass of 145
Neutral loss in MS/MS

11. iTRAQ components
• Components of iTRAQ multiplexed isobaric tagging
chemistry
1) Reporter group based on N,N-dimethylpiperazine
2) Mass balance carbonyl group
3) A peptide-reactive group (ester of N-
hydroxysuccinimide,NHS)
The m/z value of the reporter group ranges from 114.1 –
117.1.
The balance group mass is 28 – 31 Da.
The overall mass of reporter plus balance components
remains constant. 145.1 Da for all four reagents.

12. iTRAQ Experiment - Procedure
1) Protein reduction and cysteine blocking
• Dissolve protein sample in o.5M triethyl
ammoniumbicarbonate, pH 8.5
• Reduction step by adding a reducing agent
• Incubate samples at 60° C for 1H
• Block cysteine by adding a Cysteine blocking reagent
• Add trypsin solution
• Incubate overnight at 37°C
• Clean up samples using zip tip

13. 2) Labelling
Reconstitute the iTRAQ reagent in isopropanol
Add iTRAQ reagent to digested protein sample

14. 3) Combine the labelled Samples in one tube

15. 4) Purification
Pooled samples are purified on a strong cation
exchange column to remove excess unbound
reagent.
This step facilitates sample clean up.

16. Overview of protocol

17. iTRAQ – An Animation

18. iTRAQ Advantages
• Performs relative (or absolute) quantification in up
to 4 or 8 samples
• Multiplexing
• Increased analytical precision and accuracy, saves
MS run time (since isobaric labels)
• Expanded coverage of proteome by tagging tryptic
peptides
• Eliminates limitation of ICAT for dependence on
cysteine.

19. iTRAQ Disadvantages
• Possible errors in quantitation due to
• Differences in efficiency of enzymatic digestion
• Peptide pre-fractionation step
• Variability in initial protein digestion
• Tagging is performed only after individual sample
processing is done, which leads to some variations
• Reagents are very costly
• New search algorithms and databases required

20. APPLICATIONS OF iTRAQ

21. CONCLUSION
• iTRAQ is a very straightforward technique because the
labeling chemistry works very well, and labeling efficiency is
very important for how well the quantification works.
• iTRAQ has advantages over the previous technologies, and it
is a very good method for quantitative proteomics as well as
toxicogenomics.
• iTRAQ is also more sensitive than previous methods for
protein quantification because a lot of the protein changes
that we were not able to see before and this high sensitivity
is needed to be able to see changes in low-abundance
proteins.
• Despite some of its weaknesses, iTRAQ is a powerful tool for
proteomics research. Continual improvements in its
usability and technical specifications should make iTRAQ a
must have for anyone doing proteomics.