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Title Development of kinase catalyzed biotinylation to study phosphoproteomics [electronic resource] / by Kona Arachchilage Chamara Senevirathne.
Publication Info. 2013.

Location Call No. Status Notes
 Electronic Theses and Dissertations  Electronic Resource - WSU ETD    AVAIL. ONLINE
Description 299 p. : ill.
Note Advisor: Mary Kay Pflum.
Thesis Thesis (Ph.D.) -- Wayne State University, 2013.
Summary Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to identify phosphoproteins in normal and diseased states is critical to fully characterize cell biology. Our lab recently discovered kinase-catalyzed biotinylation, where ATP-biotin is utilized by kinases to label phosphopeptides or phosphoproteins with a biotin tag. To explore kinase-catalyzed biotinylation, kinetic measurements were obtained with various kinases and the data indicated that kinase-catalyzed biotinylation occurs with catalytic efficiency appropriate for phosphoproteomics application. Next, the susceptibility of the biotin tag to phosphatases was characterized and found that the phosphorylbiotin group was relatively insensitive to protein phosphatases. Importantly, robust kinase-catalyzed biotinylation occurs without the need for phosphatase inhibitor treatment. The results suggest that kinase-catalyzed biotinylation is well suited for phosphoproteomics studies, with particular utility towards monitoring low abundance phosphoproteins. Finally, we report application of kinase-catalyzed biotinylation to the enrichment of the phosphopeptides in cell lysates for mass spectrometry-based phosphoproteomics analysis. Significantly, the biotinylation strategy was used to enrich full-length phosphoprotein, which is challenging using metal ion purification. With the utility of ATP-biotin labeling for phosphoproteomics analysis established, kinase-catalyzed biotinylation can be applied to characterizing and understanding of the role of phosphorylation in various biological events.
Subject Chemistry
Added Title Wayne State University thesis (Ph.D.) : Chemistry (Organic)
OCLC # 862744652
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