Quantitative Analysis of Proteomes and Subproteomes by Isotope-Coded Affinity Tag and Solid-Phase Glycoprotein Capture
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Chemical probes for isolating specific subsets of a proteome in conjunction with mass spectrometry have had a profound influence on quantitative analysis of complex protein mixtures. Because of the dynamic range of protein abundance, comprehensive profiling of complex proteomes has been an exceedingly challenging analytical problem. However, selective isolation of a subset of proteins (i.e., a protein class) from a proteome via chemistries selective for moieties such as phosphates or sulfhydryls substantially reduces the sample complexity by one or two orders of magnitude and enriches a subclass of the proteome prior to mass spectrometric analysis (1). In this chapter, two commonly used chemical probes for selective isolation of cysteine-containing and N-linked carbohydrate-containing peptides for the quantitative analysis of a proteome are described (2, 3, 4, 5). The first is based on stable isotope affinity tagging of the cysteine residues in a protein; i.e., the original isotope-coded affinity tag (ICAT) method. The second method uses specific chemical probes that selectively isolate N-glycosylated proteins (i.e., the glycopeptide capture method) and subsequently labels the amino groups with light (d0, contains no deuteriums) or heavy (d4, contains four deteriums) forms of succinic anhydride for quantitative measurement.
KeywordsSuccinic Anhydride Sodium Periodate Selective Isolation Coupling Buffer Label Buffer
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