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Research Report, May 2002

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In a technical report in the May issue of Nature Biotechnology, Ruedi Aebersold and his colleagues at the Institute for Systems Biology in Seattle describe a solid-phase capture and release technique for labeling and isolating cysteinyl peptides. The new technique, which uses microcapillary liquid chromatography and tandem mass spectrometry to identify and compare relative quantities of the peptides, is simpler, more efficient, and more sensitive than the ICAT reagent technology, the authors claim.

To prove this, Aebersold’s team compared the solid-phase approach with the ICAT reagent technology in an analysis of galactose-induced protein expression changes in yeast. The group compared the two techniques using a larger, 100 microgram sample load, and a smaller, 10 microgram sample. In both cases, the solid phase approach identified and quantified a greater number of proteins in comparison to the ICAT reagent technology. Specifically, using the solid-phase approach, Aebersold’s team identified four proteins associated with galactose utilization, whereas the ICAT approach identified only one.

The solid-phase method is faster and simpler than the ICAT technology because isolating and labeling the cysteine-containing peptides can be accomplished in one step, the authors claim. In addition, because the peptides are covalently bound to glass beads by a photocleavable linker, the solid-phase method allows more stringent wash conditions to be used when removing the peptides not bound to the beads. In contrast, the ICAT reagent technology requires an additional step to remove the non-covalently associated molecules. The solid-phase is also potentially more versatile because it allows the use of a range of stable isotope tags, permitting more than two samples to be analyzed at once. Furthermore, because the group employed a smaller mass tag with the solid-phase method, it was less likely to fragment when analyzed using tandem mass spectrometry, thereby simplifying the resulting spectra.

However, the authors note in the paper that the ICAT approach is preferable when separating proteins using 2D gel electrophoresis, because the solid-phase approach requires that peptides be labeled only after proteolysis.

 

Researchers at the Rudbeck Laboratory and the Ludwig Institute for Cancer Research in Uppsala, Sweden, have developed a new technique for in vitro detection of proteins in quantities as low as 40 zeptomoles (40 x 10-21 mol), the researchers report in the May issue of Nature Biotechnology. The technique, termed proximity ligation, relies on pairs of DNA aptamer affinity probes, which contain sequence extensions that hybridize to a third oligonucleotide upon recognition of a target protein. The researchers use PCR to amplify the ligation product, giving information on both the presence of the protein and its quantity in the sample. The authors, led by corresponding author Ulf Landegren of the department of genetics and pathology at the Rudbeck Laboratory, claim that the technique could be extended for use with other types of binding agents, including monoclonal antibodies and affinity agents generated using phage display. In addition, the group claims that the technique should in theory allow for the detection of large sets of proteins simultaneously, by tethering different reporter DNA molecules to a standard oligonucleotide array.

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