ICAT

schematic sequence of the ICAT procedure

ICAT (from English isotope-coded affinity tag 'isotope -coded affinity tag ') is a biochemical method for quantitative protein analysis , which is based on a mass spectrometric analysis of a protein based on its tag with a protein tag , which was previously coupled with different isotopes .

properties

The samples to be examined usually consist of three fundamental elements: a functional group that is able to mark a specific side chain of an amino acid (e.g. iodoacetamide to mark cysteine residues ), a crosslinker surrounded by isotopes and a tag (e.g. . Biotin ) for the affinity- based separation of labeled proteins or peptides . To compare two proteomes quantitatively, one sample is labeled with the light version of the isotope and the other with the heaviness. To minimize errors, the substances to be examined are digested with protease (mostly trypsin ) and subjected to avidin affinity chromatography in order to isolate the isotope-coated peptides. These peptides are then analyzed by LC-MS. The ratio of the signal strengths of the various peptides which have been labeled with isotopes of different weights is used to determine the relative amount. Originally, ICAT was developed with deuterium ; due to interactions with the stationary phase , it was later switched to ¹³ C.

Individual evidence

^ SP Gygi, B. Rist, SA Gerber, F. Turecek, MH Gelb, Ruedi Aebersold : Quantitative analysis of complex protein mixtures using isotope-coded affinity tags . In: Nature Biotechnology . 17, No. 10, October 1999, pp. 994-9. doi : 10.1038 / 13690 . PMID 10504701 .
↑ US Patent 6670194: "Rapid quantitative analysis of proteins or protein function in complex Mixtures," Rudolf Hans Aebersold et al.
^ Friedrich Lottspeich , J. Kellermann: ICPL labeling strategies for proteome research. In: J. Methods Mol Biol. (2011), Volume 753, pp. 55-64. doi : 10.1007 / 978-1-61779-148-2_4 . PMID 21604115 .
↑ EC Yi, et al .: Increased quantitative proteome coverage with (13) C / (12) C-based, acid-cleavable isotope-coded affinity tag reagent and modified data acquisition scheme . In: Proteomics . 5, No. 2, 2005, pp. 380-7. doi : 10.1002 / pmic.200400970 . PMID 15648049 .

[pmid10504701-1] SP Gygi, B. Rist, SA Gerber, F. Turecek, MH Gelb, Ruedi Aebersold : Quantitative analysis of complex protein mixtures using isotope-coded affinity tags . In: Nature Biotechnology . 17, No. 10, October 1999, pp. 994-9. doi : 10.1038 / 13690 . PMID 10504701 .

[2] US Patent 6670194: "Rapid quantitative analysis of proteins or protein function in complex Mixtures," Rudolf Hans Aebersold et al.

[3] Friedrich Lottspeich , J. Kellermann: ICPL labeling strategies for proteome research. In: J. Methods Mol Biol. (2011), Volume 753, pp. 55-64. doi : 10.1007 / 978-1-61779-148-2_4 . PMID 21604115 .

[4] EC Yi, et al .: Increased quantitative proteome coverage with (13) C / (12) C-based, acid-cleavable isotope-coded affinity tag reagent and modified data acquisition scheme . In: Proteomics . 5, No. 2, 2005, pp. 380-7. doi : 10.1002 / pmic.200400970 . PMID 15648049 .