This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein expression profiling to identify global patterns of gene expression is time and labor intensive and generally allows only the 1,000 to 2,000 most abundant proteins to be analyzed unless prefractionation is used. Another approach for monitoring the physiological state of a cell is to query the status of the signal transduction pathways. One exciting way to approach this is to monitor the phosphorylation status of proteins, their sites of phosphorylation, and their changes in phosphorylation level. This has been termed phosphoproteomics by Chait (Oda, et al., 2001) and two general experimental approaches have been developed, the first based on phosphoramidate chemistry (Zhou, et al., 2001) and the second based on various Beta-elimination/Michael addition strategies (Molloy and Andrews, 2001; Oda, et al., 2001; Goshe, et al., 2002). Both require selective tagging of phosphorylated proteins or peptides followed by enrichment and analysis by mass spectrometry. Both approaches are amenable to isotope tagging for quantification. We have developed a single-pot reaction that allows proteins phosphorylated on serine and threonine residues to be selectively tagged (Molloy and Andrews, 2001). This approach relies on selective beta-elimination and Michael addition of the tag and has been used to identify the phosphorylation sites in proteins (Molloy and Andrews, 2001).
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