title: Proteome-wide discovery and characterizations of nucleotide-binding proteins with affinity-labeled chemical probes.
abstract: Nucleotide-binding proteins play pivotal roles in many cellular processes including cell signaling. However, targeted studies of the subproteome of nucleotide-binding proteins, especially protein kinases and GTP-binding proteins, remain challenging. Here, we report a general strategy in using affinity-labeled chemical probes to enrich, identify, and quantify ATP- and GTP-binding proteins in the entire human proteome. Our results revealed that the ATP/GTP affinity probes facilitated the identification of 100 GTP-binding proteins and 206 kinases with the use of low milligram quantities of lysate of HL-60 cells. In combination with the use of the stable isotope labeling by amino acids in cell culture-based quantitative proteomics method, we assessed the ATP/GTP binding selectivities of nucleotide-binding proteins at the global proteome scale. Our results confirmed known and, more importantly, unveiled new ATP/GTP-binding preferences of hundreds of nucleotide-binding proteins. Additionally, our strategy led to the identification of three and one unique nucleotide-binding motifs for kinases and GTP-binding proteins, respectively, and the characterizations of the nucleotide-binding selectivities of individual motifs. Our strategy for capturing and characterizing ATP/GTP-binding proteins should be generally applicable for those proteins that can interact with other nucleotides.
authors: Department of Chemistry, University of California, Riverside, California 92521-0403, United States.
authors: Yongsheng
authors: Y
authors: Xiao
authors: Lei
authors: L
authors: Guo
authors: Xinning
authors: X
authors: Jiang
authors: Yinsheng
authors: Y
authors: Wang
mesh_terms: D000345:Affinity Labels / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D018922:HL-60 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D010865:Pilot Projects; D017433:Protein Structure, Secondary; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D002352:Carrier Proteins; D009711:Nucleotides; D020543:Proteome