Tuesday, March 10, 2009

Absolute Quantification of Specific Proteins in Complex Mixtures Using Visible Isotope-Coded

This article illustrate to us the use of a novel type of protein tagging reagent, that the authors call , “the visible isotope-coded affinity tag” (VICAT) which permits the quantification of the absolute amount of a target protein/s within a highly complex biological sample such as a eukaryotic cell lysate. This method can tag the thiol groups of cysteines or thioacetylated amino groups and also can introduce a biotin affinity handle (a visible moiety ) which allows tracking of the chromatographic position of the target peptide, without the need of a mass spectrometrer. A photocleavable linker (for tag removal) and an isotopic tag are also introduced, which enables distinguishing between the sample and reference peptides. The authors show the application of VICAT reagents to determine the absolute abundance of human group V phospholipase A2, in eukaryotic cell lysates;combining isolectric focusing of peptides on immobilized gel strip followed by microliquid chromatography/electrospray ionization mass spectrometry, they show that 66 fmol of phospholipase A2 per 100 ugs of cell protein are found in human lung macrophages. On the other hand Western blotting did not provide conclusive results. It is envisioned that VICAT reagents may be helpful for various applications including but not limited to the analysis of lead disease markers that could be detected in serum samples.

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