Quantification of Proteins by iTRAQ - LC-MS/MS in Proteomics: Methods and Applications - page 203

Chapter 12

Quantification of Proteins by iTRAQ

Richard D. Unwin

Abstract

Protein relative quantification is a key facet of many proteomics experiments. Several methods exist for
this type of work, some of which are described elsewhere in this volume. In this chapter we will describe
the use of isobaric tags for relative and absolute quantification (iTRAQ). These chemical tags attach
to all peptides in a protein digest via free amines at the peptide N-terminus and on the side chain of
lysine residues. Labelled samples are then pooled and analysed simultaneously. Since the tags are isobaric,
labelled peptides do not show a mass shift in MS, instead signal from the same peptide from all samples
is summed, providing a moderate increase in sensitivity. Upon peptide fragmentation, sequence ions
(b- and y-type) also show this summed intensity which aids sensitivity. However, the distribution of
isotopes in the different tags is such that when the tags fragment a tag-specific 'reporter' ion is released.
The ratio of signal intensities from these tags acts as an indication of the relative proportions of that
peptide between the different labelled samples. This chapter will describe the procedure for labelling and
analysing peptide/protein samples using iTRAQ.

Key words: Peptide, protein, iTRAQ, isobaric, relative quantitation, liquid chromatography, mass
spectrometry.

1. Introduction

The use of isobaric tags is now a widely utilised strategy for
obtaining relative quantification of peptides in up to eight sam-
ples simultaneously. The principle of using isobaric tags is an
elegant one. Samples are derivatised with one of several tags,
all of which have identical overall mass but which vary in terms
of the distribution of heavy isotopes around their structure
( Fig. 12.1A ) . This means that when samples are pooled and