gygi_ICAT - 1999 Nature America Inc....

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994 NATURE BIOTECHNOLOGY VOL 17 OCTOBER 1999 RESEARCH With the completion of an increasing number of genomic sequences, attention is currently focused on how the data contained in sequence databases might be interpreted in terms of the structure, function, and control of biological systems. Approaches for global profiling of gene expression at the mRNA level as a function of the cellular state have been developed 1–3 and are widely used to identify clusters of genes for which the expression is idiotypic for a specific state. These methods, though exquisitely sensitive, do not indicate changes in protein expression. Quantitative proteome analysis, the global analysis of protein expression, is a complementary method to study steady-state gene expression and perturbation-induced changes. In comparison to gene expression analysis at the mRNA level, proteome analysis provides more accurate information about biological systems and pathways because the measurement directly focuses on the actual biological effector molecules. Most approaches to quantitative protein analysis are accom- plished by combining protein separation, most commonly by high- resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), with mass spectrometry (MS)-based or tandem mass spectrometry (MS/MS)-based sequence identification of selected, separated protein species 4–8 . This method is sequential, labor inten- sive, and difficult to automate. In addition, it selects against specif- ic classes of proteins, such as membrane proteins, very large and small proteins, and extremely acidic or basic proteins. However, the technique’s most significant flaw lies in its bias toward highly abun- dant proteins, as lower abundance regulatory proteins (e.g., tran- scription factors, protein kinases) are rarely detected when total cell lysates are analyzed 4–8 . The development of methods and instrumentation for automat- ed, data-dependent electrospray ionization (ESI) MS/MS, in con- junction with microcapillary liquid chromatography ( μ LC) and database searching, has significantly increased the sensitivity and speed for the identification of gel-separated proteins. Moreover, μ LC-MS/MS has also been used successfully for the large-scale iden- tification of proteins directly from mixtures without gel elec- trophoretic separation 9,10 . These analyses, though fast and easily auto- mated, are not quantitative and are also incompatible with the analy- sis of low-abundance proteins. Thus, there is a great need for a gener- al and quantitative technology for proteome analysis. We describe an approach to quantitative proteome analysis based on a class of reagents termed isotope-coded affinity tags (ICAT), which consist of three functional elements: a specific chemical reac- tivity, an isotopically coded linker, and an affinity tag.
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This note was uploaded on 05/28/2010 for the course WE BIBI010000 taught by Professor Marnikvuylsteke during the Spring '10 term at Ghent University.

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gygi_ICAT - 1999 Nature America Inc....

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