B

2 copies of target protein label cysteines with light (d0) ICAT

label cysteines with heavy (d$) ICAT

Icat Label Spectrum

full scan spectrum indicates LIH ratio

MS-MS spectrum indicates peptide sequence relative quantitation identification

Fig. 5. Schematic representation of relative quantitation of protein in two samples with thiol-reactive ICAT reagents and LC-MS-MS.

For example, analysis of yeast extracts yielded two ICAT-tagged peptides HHIPFYEVDLC*DR and DC*VTLK (the * indicates the ICAT-modified cysteine residue), which both mapped to the protein GAL10. Comparison of the levels of d0- and d8-ICAT-HHIPFYEVDLC*DR thus indicates the relative levels of the protein GAL10 in the two samples. This is illustrated by the comparison of proteomes of untreated yeast and yeast treated with ethanol or galactose, which induces significant changes in the levels of many enzymes of intermediary metabolism. Treatment of yeast with galactose and ethanol yielded two samples that were compared by ICAT LC-MS-MS. The ethanol-treated samples were labeled with the d0-ICAT and the galactose-treated samples were treated with the d8-ICAT. The ratio of the d0- to d8-ICAT-tagged HHIPFYEVDLC*DR and the d0- to d8-ICAT-tagged DC*VTLK was 1:>200, which indicated a greater than 200-fold elevation in GAL10 in galactose-treated yeast compared to ethanol-treated yeast.

In general, one to three representative peptides from each protein are analyzed and detected by the ICAT method. The detection of multiple peptides from a protein increases confidence in the assignment of protein identity. Moreover, multiple measurements of d0/d8 ICAT peptide ratios increase the accuracy of measurement of the relative levels of that protein in the two samples.

The ultimate expression of the ICAT approach is the combination of isotope tagging with multidimensional peptide separations prior to LC-MS-MS. As discussed in the previous chapter, the use of multidimensional protein and peptide separations combined with LC-MS-MS enhances the detection of relatively low-abundance proteins by "spreading out" the peptide mixture. This provides the MS instrument the opportunity to obtain MS-MS spectra on the greatest number of peptides in the sample. Use of multidimensional peptide separations together with isotope tagging should provide the greatest opportunity to compare changes in expression of low-abundance proteins.

The ICAT approach to proteome comparisons certainly offers some advantages over the 2D gel/MALDI-TOF-based approach. First, the use of LC-MS-MS offers more definitive identification of proteins from complex mixtures than does MALDI-TOF. Second, LC-MS-MS, particularly with multidimensional peptide separations, offers enhanced detection of low-abundance proteins compared to 2D gel analyses, which are limited by the poor dynamic range for protein staining.

Nevertheless there are some limitations of the ICAT technique. First, some proteins either do not contain cysteine residues or else they contain cysteines that are not accessible to the ICAT reagent under the conditions used for tagging. These will not be detected by the ICAT approach. Second, the ICAT approach is essentially a tool for comparing levels of protein expression in two samples. Because only peptides containing ICAT-reactive cysteines are detected in these analyses, most peptides from the proteins are "thrown away" in the avidin bead wash step. With these peptides goes much of the information about changes in protein modifications (e.g., phosphorylation) that may account for changes in the function of that protein between two samples. Unless the modification happens to occur on a cysteine-containing, ICAT-reactive peptide, it will not be detected.

Further variations of the isotope-tagging approach are likely to emerge in the near future. The need for quantitative comparisons of proteomes will become increasingly important in understanding cellular biochemistry. The generic approach is to tag the peptides in two samples with differently labeled tags, then analyze the sample and compare the levels of the differently tagged versions of each peptide. Although the ICAT approach is directed at tagging thiols, it is possible to tag other functional groups in peptides, such as N-terminal amines. This would sacrifice the strategy of greatly simplifying a complex peptide mixture and would certainly necessitate modification of the ICAT approach described earlier. Creative application of isotope-tagging strategies certainly holds great promise for quantitative proteomics.

Suggested Reading

Binz, P. A., Muller, M., Walther, D., Bienvenut, W. V., Gras, R., Hoogland, C., et al. (1999) A molecular scanner to automate proteomic research and to display proteome images. Anal. Chem. 71, 4981-4988. Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994-999. Lemkin, P. F. (1997) Comparing two-dimensional electrophoretic gel images across the Internet. Electrophoresis 18, 461-470. Wilkins, M. R., Gasteiger, E., Bairoch, A., Sanchez, J. C., Williams, K. L., Appel, R. D., and Hochstrasser, D. F. (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol. Biol. 112, 531-552.

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