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Identification of multiple metabolic enzymes from mice cochleae tissue using a novel functional proteomics technology.

Wang DL, Li H, Liang R, Bao J - PLoS ONE (2015)

Bottom Line: The technology combines the ability of two-dimensional gel electrophoresis (2-DE) to separate proteins with a protein elution plate (PEP) to recover active proteins for functional analysis and mass spectrometry (MS)-based identification.Using MS, five NADH-dependent oxidases were identified that showed highly altered enzymatic activities due to the drug treatment.In essence, the PEP technology allows for a systematic analysis of a large enzyme family from a complex proteome, providing insights in understanding the mechanism of drug treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Vanderbilt University, Nashville, TN, United States of America.

ABSTRACT
A new type of technology in proteomics was developed in order to separate a complex protein mixture and analyze protein functions systematically. The technology combines the ability of two-dimensional gel electrophoresis (2-DE) to separate proteins with a protein elution plate (PEP) to recover active proteins for functional analysis and mass spectrometry (MS)-based identification. In order to demonstrate the feasibility of this functional proteomics approach, NADH and NADPH-dependent oxidases, major redox enzyme families, were identified from mice cochlear tissue after a specific drug treatment. By comparing the enzymatic activity between mice that were treated with a drug and a control group significant changes were observed. Using MS, five NADH-dependent oxidases were identified that showed highly altered enzymatic activities due to the drug treatment. In essence, the PEP technology allows for a systematic analysis of a large enzyme family from a complex proteome, providing insights in understanding the mechanism of drug treatment.

No MeSH data available.


Diagram of the PEP Technology with MS protein identification.
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pone.0121826.g001: Diagram of the PEP Technology with MS protein identification.

Mentions: After functional protein assays were carried out in separate 384-well microplates (Fig. 1), the next step in the development of the PEP was to demonstrate if proteins transferred to the Master Plate could be successfully transferred to enzyme assay plates for functional assays. In the current setting, protein mixtures at levels of 10 ng/well, 100 ng/well and 1,000 ng/well were tested for recovery from the master plate after an incubation of 4 hours. It was found that a significant amount of proteins could be recovered from the Master Plate, especially at levels typically seen in the 2-DE gel (100 ng/well-1,000 ng/well). Given the current detection sensitivity of mass spec technology, this should be suitable for protein characterization and identification.


Identification of multiple metabolic enzymes from mice cochleae tissue using a novel functional proteomics technology.

Wang DL, Li H, Liang R, Bao J - PLoS ONE (2015)

Diagram of the PEP Technology with MS protein identification.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4374962&req=5

pone.0121826.g001: Diagram of the PEP Technology with MS protein identification.
Mentions: After functional protein assays were carried out in separate 384-well microplates (Fig. 1), the next step in the development of the PEP was to demonstrate if proteins transferred to the Master Plate could be successfully transferred to enzyme assay plates for functional assays. In the current setting, protein mixtures at levels of 10 ng/well, 100 ng/well and 1,000 ng/well were tested for recovery from the master plate after an incubation of 4 hours. It was found that a significant amount of proteins could be recovered from the Master Plate, especially at levels typically seen in the 2-DE gel (100 ng/well-1,000 ng/well). Given the current detection sensitivity of mass spec technology, this should be suitable for protein characterization and identification.

Bottom Line: The technology combines the ability of two-dimensional gel electrophoresis (2-DE) to separate proteins with a protein elution plate (PEP) to recover active proteins for functional analysis and mass spectrometry (MS)-based identification.Using MS, five NADH-dependent oxidases were identified that showed highly altered enzymatic activities due to the drug treatment.In essence, the PEP technology allows for a systematic analysis of a large enzyme family from a complex proteome, providing insights in understanding the mechanism of drug treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Vanderbilt University, Nashville, TN, United States of America.

ABSTRACT
A new type of technology in proteomics was developed in order to separate a complex protein mixture and analyze protein functions systematically. The technology combines the ability of two-dimensional gel electrophoresis (2-DE) to separate proteins with a protein elution plate (PEP) to recover active proteins for functional analysis and mass spectrometry (MS)-based identification. In order to demonstrate the feasibility of this functional proteomics approach, NADH and NADPH-dependent oxidases, major redox enzyme families, were identified from mice cochlear tissue after a specific drug treatment. By comparing the enzymatic activity between mice that were treated with a drug and a control group significant changes were observed. Using MS, five NADH-dependent oxidases were identified that showed highly altered enzymatic activities due to the drug treatment. In essence, the PEP technology allows for a systematic analysis of a large enzyme family from a complex proteome, providing insights in understanding the mechanism of drug treatment.

No MeSH data available.