iTRAQ

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In iTRAQ ( i sobaric T ags for R elative and A m pletely Q uantitation ) is an experimental method in the field of protein analysis and proteomics . It is used to quantify various proteins and peptides together using mass spectrometry . iTRAQ uses isobar labeling and tandem mass spectrometry , among others .

Procedure

iTRAQ kit for 8 samples

iTRAQ is used in proteomics for protein characterization, since with the method up to four, with the further development 8-Plex up to eight samples can be examined simultaneously. For the application of iTRAQ method, it is first necessary to the N-terminus and the amino - side chains present in the sample peptides having covalently bonded (hereinafter with various molecules Label or Tag ) of different molecular weights to be marked. Two different reagents are currently used for this purpose: 4-plex and 8-plex. The tags have different masses, fragment differently in the mass spectrometer and can therefore be differentiated in the mass spectrum .

Any peptides of various origins can be labeled. The samples to be examined together are then combined ( pooled ), then usually fractionated by nano HPLC and analyzed by means of tandem mass spectrometry ( MS / MS ). A subsequent database search with the fragmentation data obtained allows the peptides to be identified and the corresponding proteins to be determined.

The fragmentation of the introduced tags generates an ion with a typical mass, the so-called reporter ion , in the lower mass range, depending on the tag type used , the frequency of which can be used to determine the amount of peptides present in the same spectrum (and thus the proteins) relative to each other. It is therefore a gel-free method for relative quantification (comparative quantity determination).

If the absolute amount of one of the samples is known, the method also allows an absolute quantification, since the amount of each additional sample can be calculated using the ratio.

Data evaluation

Peptide level

Example of iTRAQ reporter in the mass spectrum, reporter circled in red on the left. The singly charged reporter ions are visible in the lower spectrum at the mass / charge ratios (m / z) 114, 115, 116 and 117.

The signals of the reporter ions in each MS / MS spectrum make it possible to calculate the relative frequency ( ratio ) of the peptides to the reporter that were identified via this spectrum. Due to measurement inaccuracies, it may well happen that the reporter ions are each represented by more than one signal in the spectrum and these have to be combined into a peak in a suitable manner and without information corruption. For example, to summarize the intensity of several peaks, there are two possible approaches:

Integrating the area of ​​the spectrum here leads to larger errors if the spacing of the peaks and the number of peaks are not the same. 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 known amount.

software

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

  • i-Tracker , a simple program that calculates a trapezoidal area of ​​constant width around the peaks and uses this to determine the ratios.
  • Quant , a Matlab script that implements the statistically precise algorithm.
  • Quant for windows , the Windows implementation of Quant.
  • jTraqX , Quant's portable Java implementation.

Remarks

  1. ↑ Download from sourceforge.net
  2. ↑ Download from sourceforge.net
  3. ↑ Download from sourceforge.net

Individual evidence

  1. ↑ 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: Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents . In: Mol. Cell. Proteomics . 3, No. 12, 2004, pp. 1154-1169. doi : 10.1074 / mcp.M400129-MCP200 . PMID 15385600 .
  2. ↑ Zieske LR: A perspective on the use of iTRAQ reagent technology for protein complex and profiling studies . In: J. Exp Bot.. . 57, No. 7, 2006, pp. 1501-1508. doi : 10.1093 / jxb / erj168 . PMID 16574745 .
  3. ↑ Gafken PR, Lamp PD: Methodologies for characterizing phosphoproteins by mass spectrometry . In: Cell Commun. Adhes. . 13, No. 5-6, 2006, pp. 249-262. doi : 10.1080 / 15419060601077917 . PMID 17162667 . PMC 2185548 (free full text).
  4. ↑ Choe L, D'Ascenzo M, Relkin NR, Pappin D, Ross P, Williamson B, Guertin S, Pribil P, Lee KH: 8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease . In: Proteomics . 7, No. 20, 2007, pp. 3651-60. doi : 10.1002 / pmic.200700316 . PMID 17880003 .
  5. ↑ a b c d Boehm, AM, Puetz, S., Altenhofer, D., Sickmann, A., Falk, M., Precise protein quantification based on peptide quantification using iTRAQ , BMC Bioinformatics, 2007, 8: p. 214, doi : 10.1186 / 1471-2105-8-214 .
  6. ↑ Shadforth IP, Dunnley PJ, Lilley KS, Bessant C: i-Tracker: For quantitative proteomics using iTRAQ . In: BMC Genomics . 6, 2005, p. 145. doi : 10.1186 / 1471-2164-6-145 . PMID 16242023 . PMC 1276793 (free full text).
  7. ↑ Boehm, AM, Altenhöfer, D., Pütz, S., Precise and Statistically Sound Protein Quantification in Mass Spectrometry Based Proteomics Using iTRAQ, in: Küng, J., Schneider, K., Wagner, R., 2nd International Conference on Bioinformatics Research and Development (BIRD'08), series Informatik, 26, Trauner Verlag, Linz, 2008: pp. 3–12.
  8. ↑ Muth, T., Keller, D., Puetz, SM, Martens, L., Sickmann, A., Boehm, AM, jTraqX: a Free, Platform Independent Tool for Isobaric Tag Quantitation at the Protein Level , Proteomics, 2010, 10 (6): pp. 1223-1225, doi : 10.1002 / pmic.200900374 .