Recent advances in protein interaction study, new techniques.Help in detection of biomarker (protein) for human disease in single experiment.

1. iTRAQ Protein labeling technique
Presented by
Bharti Rakhecha

2. Contents
Introduction
Objective of developing iTRAQ
Principle of iTRAQ & its Mechanism
Advantage & Disadvantage
References

3. Introduction
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.

4. iTRAQ technology for
protein quantitation using
mass spectrometry
Current identification
software the ProQuant
software
i-Tracker software has
been developed to extract
reporter ion peak ratios
from non-centroided
tandem MS peak lists
protein identification
tools such as Mascot and
Sequest.
Four-fold multiplexing
reaction

5. Objective of developing iTRAQ
Many differential
effects on proteins
themselves come
from post-
translational
modifications.
Protein
expression
cannot be
measured or
identified by
looking at the
mRNA levels.
By studying
effector molecules
will contribute to
better
understanding of
disease & in
developing new
treatment.

6. ( Journal of proteome research, Rauniyar N et al (2014))

7. Principle
Isobaric mass tags have identical overall mass but vary in terms of
the distribution of heavy isotopes around their structure.
Most common isobaric tag is amine-reactive
Tags employ N-hydroxysuccinimide (NHS) chemistry
an amine-
reactive
group
isotopic
reporter
group
isotopic
balance
group

8. Binding of Isobaric Tags
The amine-reactive, NHS-ester-activated group reacts with N-
terminal amine groups and ε- amine groups of lysine residues
to attach the tags to the peptides.
The labeling is efficient for all peptides regardless of protein
sequence or proteolytic enzyme specificity.
The labeling does not occur, however, if the primary amino
groups are modified, such as when N-terminal glutamine or
glutamic acid forms a ring (pyro-glutamic acid) or if the group
is acetylated.
For successful quantification, labeling should be specific to the
targeted residues (N-terminal amine and lysyl ε-amine groups
in a peptide) and should proceed to completion.

9. ( Journal of proteome research, Rauniyar N et al (2014)

10. i
T
R
A
Q
Overall mass of the reporter and balance components of
the molecule are kept constant using differential isotopic
enrichment with 13C,15N, and18O atoms.
Reporter group ranges in mass from m/z114−117, whereas
the balance group ranges in mass from 28 to 31 Da.
Reporter groups of the iTRAQ reagents will split from the
peptide and form small fragments with mass/charges
(m/z) of 114, 115, 116, and 117.
Intensity of each of these peaks represents quantity
of small reporter group fragment and thus represents
the quantity of a peptide sample.

11. Data Analysis
(www.intechopen.com)

12. Factors affecting
iTRAQ
Evaluation of Labeling Efficiency and Isotope
Impurity Correction
Ratio Compression and Its Correction
Reporter Ion Intensity Dynamic Range
Effect of Unique and Shared Peptides in
Inferring Protein Ratios
Estimation of Protein Fold Changes
Comparison of Multiple Isobaric Labeling
Experiments

13. Advantage & Disadvantage
Advantage
High throughput
quantification
Ability to combine &
analyze several sample
in one experiment
Reduce overall
time &
variation
Statistical
Validation
Studying protein
interaction & pattern
of expression
No interference
with peptide
fragmentation
Improve
efficiency of
MS/MS
fragmentation

14. Disadvantage
Very sensitive to
contamination
from salts
Extremely
costly
Variability arise
due to inefficient
enzymatic
digestion
Sophisticated
software
required

15. References
1. Niu R., Liu Y., Zhang Y., Wang H., Wang Y., Wang W., Li X (2017). iTRAQ-Based
Proteomics Reveals Novel Biomarkers for Idiopathic Pulmonary Fibrosis. PLoS ONE 12(1):
e0170741.
2. Rauniyar N., Yates JR (2014). Isobaric Labeling-Based Relative Quantification in
Shotgun Proteomics. J. Proteome 13: 1529-5309.
3. Shadforth IP., Dunkley T., Lilley K., Bessant C (2005). i-Tracker: For quantitative
proteomics using iTRAQ. BMC Genomics1471-2164/6/145.
4. Linke D., Hung CW., Cassidy L., Tholey A (2013).Optimized fragmentation conditions
for iTRAQ- labeled phosphopeptides. J. Proteome Res 12: 2755−2763.
5. Herbrich Sm., Cole RN., West KP., Sahulze K., Yager JD., Groopman JD., Christian P., Wu
L., O’ Meally RN., May DH., Mcintosh MW., Ruczinski I(2013). Statistical inference from
multiple iTRAQ experiments without using common reference standards. J. Proteome
Res.12: 594−604.
6. Ross PL., Huang YN., Marchese JN., Williamson B., Parker K., Hattan S., Khainovski N.,
Pillai S., Dey S., Daniels S., Purkayastha S., Juhasz P., Martin S., Bartlet-Jones M., He F.,
Jacobson A., Pappin DJ (2004). "Multiplexed protein quantitation in Saccharomyces
cerevisiae using amine-reactive isobaric tagging reagents". Mol. Cell. Proteomics. 3 (12):
1154–69.
7. Zieske LR (2006). "A perspective on the use of iTRAQ reagent technology for protein
complex and profiling studies". J. Exp. Bot. 57 (7): 1501–8.