iTRAQ® (isobaric tags for relative and absolute quantitation) method

iTRAQ® allows global analyses of protein expression and quantitative comparison among samples by mass spectrometry.

Why global analyses of protein expression are needed

Principle of the iTRAQ® method

The structure of the iTRAQ® reagent is shown in Figure 1. This reagent consists of an active ester (NHS) moiety and an isobaric tag (mass: 305) for reaction with the N-terminal amino groups of peptides and the amino groups of lysine. Furthermore, the isobaric tag consists of a balance group and a reporter group. There are eight kinds of iTRAQ® reagents, each having stable isotopes that are uniquely distributed between the balance and reporter groups. Identical peptides obtained from different samples can be labeled with different iTRAQ® reagents to be used as iTRAQ®-modified peptides having the same mass (Figure 2). When iTRAQ®-modified peptides are analyzed by MS/MS, the ratios of peptide quantities among different samples are expressed as signal intensity ratios of the reporter groups (m/z: 113, 114, 115, 116, 117, 118, 119, and 121). After 1) denaturation, reduction and alkylation, 2) enzyme digestion, and 3) iTRAQ® modification, proteins in different samples are mixed to be analyzed by LC-MS/MS (Figure 3). Identical peptides derived from different samples have the same mass. However, in MS/MS analysis, the signal intensity ratios of the reporter groups indicate the ratios of the peptide quantities and can be used to determine the relative quantities of the peptides.

Figure 1

Summary of the experimental protocol for the iTRAQ(r) method

After denaturation, reduction, alkylation, and digestion, each protein sample is modified with a distinct iTRAQ(r) reagent. The iTRAQ(r)-modified samples are then mixed to be analyzed by mass spectrometry. The MS/MS spectra of the individual peptides show signals reflecting amino acid sequences and also show reporter ions reflecting the protein contents of the samples.

Figure 2

Figure 3

* iTRAQ is the trademark of Applied Biosystems, Inc.
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