Isobaric tag for relative and absolute quantitation

For iTRAQ ( isobaric Tags for Relative and Absolute Quantitation ) is an experimental method in the field of protein analysis and proteomics. It is used to quantify various common proteins and peptides by mass spectrometry. iTRAQ used inter alia Isobarenmarkierung and tandem mass spectrometry.

Method

ITRAQ used in proteomics for protein characterization using the method as up to four, with the advancement 8 -Plex may be assayed simultaneously for up to eight samples. The application of the iTRAQ technique it is first necessary, the N-terminus and the amino acid side chains present in the sample with peptides covalently linked with various molecules ( known as label or tag ) of different molecular weights to marking. At present, these are two different reagents used: 4 -plex and 8 -plex. The tags have different masses, fragmentation in the mass spectrometer different and therefore can be distinguished in the mass spectrum.

Any number of peptides of different origins are marked. Together to be tested samples are then combined ( pooled ), then typically fractionated by nano - HPLC and analyzed by tandem mass spectrometry (MS / MS). Subsequent database search using the data obtained in the fragmentation makes it possible to identify the peptides and thus to determine the corresponding proteins.

The fragmentation of the inserted tags generates in the lower mass range, depending on the tag type used in each case an ion having a typical mass, the so-called reporter ion, the frequency of occurrence may be used to the amount present in the same range of peptides ( and hence the proteins) relative to each other to be determined. It is a gel- free method for the relative quantification ( comparatively amount determination).

The absolute amount of the sample is known, the method also allows for absolute quantification, since the amount of each additional sample can be calculated using the ratio.

Data analysis

Peptide level

The signals in each of the reporter ion MS / MS spectrum make it possible to calculate the relative abundance (ratio) of the peptides for the reporter, that were identified by this range. Due to measurement inaccuracies can occur quite possible that the reporter ions are represented by more than one signal in each spectrum and these have to be combined in an appropriate way and without information revolution to a peak. To summarize, for example, the intensity of several peaks, there are two possible approaches:

  • Summing the intensities of the peaks
  • Integration

Here to integrate the area of the spectrum results in larger errors, if the distances between the peaks and the number of the peaks are not equal. The intensities of these signals should therefore be added in order to obtain a smaller error in this case.

Protein level

The ratios of the peptides are log-normally distributed. Therefore, the median of the peptide ratios of a protein represents the relative quantification of this protein compared to the reporter with a known quantity.

Software

The data of the MS / MS spectra can be analyzed with the following freely available software

  • I- Tracker, a simple program that calculates the peaks a trapezoidal surface of constant width and these are used to determine the ratios.
  • Quant [A 1], a Matlab script that implements the statistically precise algorithm.
  • Avatar for windows [A 2], the Windows implementation of Quant.
  • JTraqX [A 3], the portable Java implementation of Quant.

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