Dataset Information

A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology.

ABSTRACT: While more than a thousand protein kinases (PK) have been identified in the Arabidopsis thaliana genome, relatively little progress has been made toward identifying their individual client proteins. Herein we describe the use of a mass spectrometry-based in vitro phosphorylation strategy, termed Kinase Client assay (KiC assay), to study a targeted-aspect of signaling. A synthetic peptide library comprising 377 in vivo phosphorylation sequences from developing seed was screened using 71 recombinant A. thaliana PK. Among the initial results, we identified 23 proteins as putative clients of 17 PK. In one instance protein phosphatase inhibitor-2 (AtPPI-2) was phosphorylated at multiple-sites by three distinct PK, casein kinase1-like 10, AME3, and a Ser PK-like protein. To confirm this result, full-length recombinant AtPPI-2 was reconstituted with each of these PK. The results confirmed multiple distinct phosphorylation sites within this protein. Biochemical analyses indicate that AtPPI-2 inhibits type 1 protein phosphatase (TOPP) activity, and that the phosphorylated forms of AtPPI-2 are more potent inhibitors. Structural modeling revealed that phosphorylation of AtPPI-2 induces conformational changes that modulate TOPP binding.

SUBMITTER: Ahsan N

PROVIDER: S-EPMC3888875 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology.

Ahsan Nagib N Huang Yadong Y Tovar-Mendez Alejandro A Swatek Kirby N KN Zhang Jingfen J Miernyk Ján A JA Xu Dong D Thelen Jay J JJ

Journal of proteome research 20130115 2

While more than a thousand protein kinases (PK) have been identified in the Arabidopsis thaliana genome, relatively little progress has been made toward identifying their individual client proteins. Herein we describe the use of a mass spectrometry-based in vitro phosphorylation strategy, termed Kinase Client assay (KiC assay), to study a targeted-aspect of signaling. A synthetic peptide library comprising 377 in vivo phosphorylation sequences from developing seed was screened using 71 recombina ...[more]

PMID: 23270405

Similar Datasets

Project description:Public health threats posed by emerging respiratory infections are a significant concern, particularly in children and infants. Traditional culture-based detection methods are time-consuming and typically require 1-3 days. Herein, we developed and evaluated a 23-plex common respiratory pathogen mass spectrometry assay that enables the simultaneous detection of 18 common respiratory pathogens in children. This assay combines matrix-assisted laser desorption/ionization time of flight mass spectrometry with multiplex reverse transcription-PCR and targets 11 bacterial and 7 viral pathogens (including 10 subtypes), and two internal controls. The detection limit of the common respiratory pathogen mass spectrometry assay was as low as 1 copy/µL, with no cross-reactivity with other organisms. We assessed the clinical performance of the common respiratory pathogen mass spectrometry assay using respiratory samples from 450 children. The total 450 clinical specimens underwent analysis via matrix-assisted laser desorption/ionization time of flight mass spectrometry, and the outcomes were juxtaposed with those derived from real-time reverse-transcriptase PCR conducted concurrently. The concordance between these methods was 96.0%, and the multiple infection identification rate was 7.1%. This innovative approach enables the simultaneous analysis of numerous outcomes from a solitary examination across 192 specimens within a timeframe of approximately 7 hours, with a dramatically reduced sample use and cost. In summary, the common respiratory pathogen mass spectrometry assay is a sensitive, accurate, and cost-effective method for detecting common respiratory pathogens in children and has the potential to revolutionize the diagnosis of respiratory tract infections. IMPORTANCE This study aimed to present and evaluate a novel co-detection method that enables the simultaneous identification of 11 bacterial and 7 viral pathogens in about 7 hours using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Our approach utilizes a combination of multiplex reverse transcription-PCR and matrix-assisted laser desorption/ionization time of flight mass spectrometry, which overcomes the limitations of conventional assays, which include a long assessment time, technical difficulty, and high costs. As a screening method for common respiratory pathogens in children, common respiratory pathogen mass spectrometry assay has the potential to revolutionize the diagnosis of respiratory tract infections by providing an accurate etiological diagnosis. The common respiratory pathogen mass spectrometry assay is expected to be a critical tool for the diagnosis of respiratory infections in children, offering a more efficient, cost-effective, and accurate approach for the detection of common respiratory pathogens.

Dataset Information

A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology.

Publications

A versatile mass spectrometry-based method to both identify kinase client-relationships and characterize signaling network topology.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets