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Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria.

ABSTRACT: Protein carbonylation has been associated with various pathophysiological processes. A representative reactive carbonyl species (RCS), 4-hydroxy-2-nonenal (HNE), has been implicated specifically as a causative factor for the initiation and/or progression of various diseases. To date, however, little is known about the proteins and their modification sites susceptible to "carbonyl stress" by this RCS, especially in the liver. Using chemoprecipitation based on a solid-phase hydrazine chemistry coupled with LC-MS/MS bottom-up approach and database searching, we identified several protein-HNE adducts in isolated rat liver mitochondria upon HNE exposure. The identification of selected major protein targets, such as the ATP synthase ?-subunit, was further confirmed by immunoblotting and a gel-based approach in combination with LC-MS/MS. A network was also created based on the identified protein targets, which showed that the main protein interactions were associated with cell death, tumor morphology and drug metabolism, implicating the toxic nature of HNE in the liver mitoproteome. The functional consequence of carbonylation was illustrated by its detrimental impact on the activity of ATP synthase, a representative major mitochondrial protein target for HNE modifications.

SUBMITTER: Guo J

PROVIDER: S-EPMC3199370 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria.

Guo Jia J Prokai-Tatrai Katalin K Nguyen Vien V Rauniyar Navin N Ughy Bettina B Prokai Laszlo L

Journal of proteomics 20110723 11

Protein carbonylation has been associated with various pathophysiological processes. A representative reactive carbonyl species (RCS), 4-hydroxy-2-nonenal (HNE), has been implicated specifically as a causative factor for the initiation and/or progression of various diseases. To date, however, little is known about the proteins and their modification sites susceptible to "carbonyl stress" by this RCS, especially in the liver. Using chemoprecipitation based on a solid-phase hydrazine chemistry cou ...[more]

PMID: 21801862

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Project description:Peroxidation of cellular membrane lipids, rich in polyunsaturated fatty acids, generates electrophilic, α, β-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE is a highly reactive and cytotoxic molecule that can react with the nucleophilic sites in proteins causing posttranslational modification. The identification of protein targets is an important first step; however, quantitative profiling of site-specific modifications is necessary to understand the biological impact of HNE-induced carbonylation. We report a method that uses light (H(12)CHO) and heavy (D(13)CDO) isotopic variant of formaldehyde to differentially label primary amines (N-termini and ε-amino group of lysines) in peptides through reductive methylation and, combined with selective enrichment of modified peptides, permits comparison of the extent of carbonylation in two samples after mixing for simultaneous liquid chromatography-mass spectrometry. Specifically, dimethyl-labeled peptide carbonyls were fractionated from unmodified peptides using solid-phase hydrazide chemistry to immobilize them to porous glass beads and, after removing the unmodified peptides by thoroughly washing the beads, subsequently recover them by acid-catalyzed hydrolysis. The method was developed using HNE-modified synthetic peptides and also showing enrichment from a complex matrix of digested human plasma proteins. Applicability was confirmed using apomyoglobin as an analyte, implicating thereby its potential value to proteome-wide identification and relative quantification of posttranslational protein carbonylation with residue-specific information. Because HNE attachment may not necessarily cause change in protein abundance, this modification-focused quantification should facilitate the characterization of accompanied changes in protein function and, also, provide important insights into molecular signaling mechanisms and a better understanding of cellular processes associated with oxidative stress.

Dataset Information

Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria.

Publications

Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria.

Similar Datasets

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