UC Riverside

Nucleotide-binding proteins such as ATP- and GTP- binding proteins play pivotal roles in many cellular processes. However, targeted study of nucleotide-binding proteins, especially protein kinases and GTPases, remained challenging, which is mainly because of the lack of efficient enrichment approaches to selectively capture nucleotide-binding proteins. In this dissertation, we reported a general strategy in using affinity-labeled chemical probes to enrich, identify, and quantify ATP- and GTP- as well as other nucleotide binding proteins in the entire human proteome.

In Chapter two, we described the synthesis and application of biotin-conjugated ATP and GTP affinity probes. The ATP/GTP affinity probes facilitated the identification of 100 GTP-binding proteins and 206 kinases with the use of low mg quantities of cell lysate. Additionally, our strategy led to the identification of three and one unique nucleotide-binding motifs for kinases and GTP-binding proteins, respectively. We also assessed the ATP/GTP binding selectivities of nucleotide-binding proteins at the global proteome scale.

In Chapter three, an affinity profiling strategy to comprehensively characterize ATP-protein interactions at the entire proteome scale was developed by comparing the labeling behaviors of lysine residues with the use of low and high concentrations of the ATP affinity reagents. This novel quantitative ATP-affinity profiling strategy is particularly useful for unveiling previously unrecognized nucleotide-binding property and binding sites in whole proteome.

In Chapter four, we extended the use of ATP/GTP affinity probes and introduced an orthogonal strategy encompassing the nucleotide-affinity profiling assay and nucleotide-binding competition assay to comprehensively characterize SGTP-binding proteins along with the specific binding sites from the entire human proteome. More than 160 proteins involved in different biological processes were determined to be SGTP-binding targets.

In Chapter five, we introduced a novel global kinome profiling method, based on a newly developed isotope-coded ATP-affinity probe and a targeted proteomic method using multiple-reaction monitoring (MRM), for assessing simultaneously the expression of more than 300 kinases in human cells and tissues. This facile and accurate kinome profiling assaycould provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer chemotherapy.