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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.

SUBMITTER: Xiao Y

PROVIDER: S-EPMC3606491 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Proteome-wide discovery and characterizations of nucleotide-binding proteins with affinity-labeled chemical probes.

Xiao Yongsheng Y Guo Lei L Jiang Xinning X Wang Yinsheng Y

Analytical chemistry 20130228 6

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 identific ...[more]

PMID: 23413923

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Project description:A novel electrochemical detection approach using DNA probes labeled with Anthraquinone (AQ) as a reporter moiety has been successfully exploited as a method for the direct detection of DNA targets. This assay uses simple voltammetry techniques (Differential Pulse Voltammetry) to exploit the unique responsiveness of AQ to its chemical environments within oxygenated aqueous buffers, providing a specific detection mechanism as a result of DNA hybridization. This measurement is based on a cathodic shift of the reduction potential of the AQ tag and the concurrent reduction in peak current upon DNA binding. The further utility of this approach for discrimination of closely related DNA targets is demonstrated using DNA strands specific to B. anthracis and closely related bacillus species. DNA targets were designed to the rpoB gene incorporating nucleotide polymorphisms associated with different bacillus species. This assay was used to demonstrate that the shift in reduction potential is directly related to the homology of the target DNA. The discriminatory mechanism is dependent on the presence of oxygen in the measurement buffer and is strongly linked to the position of the nucleotide polymorphisms; with homology at the terminus carrying the AQ functionalised nucleotide critical to achieving accurate discrimination. This understanding of assay design was used to demonstrate an optimized assay capable of discriminating between Yersinia pestis (the causative agent of plague) and closely related species based on the groEL gene. This method is attractive as it can not only detect DNA binding, but can also discriminate between multiple Single Nucleotide Polymorphisms (SNPs) within that DNA without the need for any additional reagents, reporters, or processes such as melting of DNA strands. This indicates that this approach may have great potential to be exploited within novel biosensors for detection and diagnosis of infectious disease in future Point of Care (PoC) devices.

Dataset Information

Proteome-wide discovery and characterizations of nucleotide-binding proteins with affinity-labeled chemical probes.

Publications

Proteome-wide discovery and characterizations of nucleotide-binding proteins with affinity-labeled chemical probes.

Similar Datasets

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