This document provides an overview of iTRAQ techniques for protein quantification. It begins with an introduction describing iTRAQ as an isobaric labeling method used in quantitative proteomics to determine the amount of proteins from different sources. It then discusses the objective of developing iTRAQ to study protein expression and post-translational modifications. The principle section explains that iTRAQ uses tags with identical mass but varying isotope distributions to label protein peptides, allowing quantification. The technique has advantages of improved MS/MS efficiency and studying protein interactions, but also disadvantages like requiring specialized software and being sensitive to contamination.

1. “
”
iTRAQ Techniques for Quantification

2. Course Outline
 Introduction
 Objective of developing iTRAQ
 principle of iTRAQ & its Mechanism
 Advantage & Disadvantages
 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 amou nt of proteins from
different sources in a single experiment.
 It uses stable isotope labeled molecules that can be covalent bonded
to the N-termjnus and side chain amines of proteins.

4. Objective of developing iTRAQ

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.
By studying effector
molecules will
contribute to better
understanding of
disease & in
developing new
treatment.
 Many
differential
effects on
proteins
themselves
come from post
translational
modifications.

5. 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

6. Binding of Isobaric Tags
 The amine-reactive, NHS-ester-activated group reacts with Nterminal amine
groups and E- 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 occurj however, if the primary amino groups are
modified, such as when N-terminal glutamine or glutamic acid forms a ring
(pyro-gtutamic acid} or if the group is acetylated.

7. Overall mass of the reporter and balance components of the molecule
are kept constant using differential isotopic enrichment with 13C, 15N,
and 18 O atoms.
Reporter group ranges in mass from m/2114-117, whereasthe 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 .
Intensity of each of these peaks represents quantity of small reporter
group fragment and thus represents the quantity of a peptide sample.
I
T
R
A
Q

9. Advantages
Improve
efficiency of
MS/MS
fragmentatio
n
Studying
protein
interaction &
pattern of
expression

10. Disadvantages
Sophisticated
software
required
Variability
arise due to
inefficient
enzymatic
digestion
Extremely
costly
Very sensitive
to
contaminatio
n from salts

11. References
 1. Niu Liu Zhang Y., wang wang wang (2017), iTRAQ.Based
 Proteomics Reveals Novel Biomarkers for Idiopathic Pulmonary Fibrosis. PLOS ONE 1211k e0170741.
 2. Rauniyar N.. Yates JR (2014), isobaric L.abeling•8ased Relative Quantification in Shotgun Proteomics. J. Proteome 13: 1529-5309.
 Shadforth IP., Dunkley i, Lilley K., C (2005), i.Tracker; For quantitative proteomics using iTRAQ. BMC Genomics1471.2164/6/145.
 Linke D.. Hung Cassidy L.. Tholey A (20131.0ptimized fragmentation conditions for iTRAQ- labeled Proteome Res 12:2755-2763.
 Herbrich Sm.* Cole West Sahulze K.. YagerJD., Groopman JD.. Christian P., Wu L, O' Meally RN., May DI-1., Mcintosh MW„ Ruczinski
'(2013). Statistical inference from multiple iTRAQ experiments without using common reference standards." Proteome
 Res.12: 594-604.
 Ross PL., Huang YN., Marchese JN., Williamson B.. Parker K.. Hattan Khainovski N.,
 Pillai S.. Dey Sm. Daniels S.* S.. juhasz P.. Martin S.. Bartlet•jones M.r He F.* Jacobson A, Pappin DJ (200•0. "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 stud ies". J. Exp. Bot 57

12. THANK YOU