Dataset Information

Detection and identification of 4-hydroxy-2-nonenal Schiff-base adducts along with products of Michael addition using data-dependent neutral loss-driven MS3 acquisition: method evaluation through an in vitro study on cytochrome c oxidase modifications.

ABSTRACT: We report a data-dependent neutral-loss-driven MS(3) acquisition to enhance, in addition to abundant Michael adducts, the detection of Schiff-base adducts of proteins and 4-hydroxy-2-nonenal, a reactive end product of lipid peroxidation. In vitro modification of cytochrome c oxidase, a mitochondrial protein complex, was used as a model to evaluate the method. The technique allowed for a confident validation of modification sites and also identified a Schiff-base adduct in subunit Vb of the protein complex.

SUBMITTER: Rauniyar N

PROVIDER: S-EPMC3065305 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Detection and identification of 4-hydroxy-2-nonenal Schiff-base adducts along with products of Michael addition using data-dependent neutral loss-driven MS3 acquisition: method evaluation through an in vitro study on cytochrome c oxidase modifications.

Rauniyar Navin N Prokai Laszlo L

Proteomics 20091101 22

We report a data-dependent neutral-loss-driven MS(3) acquisition to enhance, in addition to abundant Michael adducts, the detection of Schiff-base adducts of proteins and 4-hydroxy-2-nonenal, a reactive end product of lipid peroxidation. In vitro modification of cytochrome c oxidase, a mitochondrial protein complex, was used as a model to evaluate the method. The technique allowed for a confident validation of modification sites and also identified a Schiff-base adduct in subunit Vb of the prote ...[more]

PMID: 19771555

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Project description:4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys(73) and, to the lesser extent, the active site Cys(32). More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys(73), whereas Cys(32) exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo.

Dataset Information

Detection and identification of 4-hydroxy-2-nonenal Schiff-base adducts along with products of Michael addition using data-dependent neutral loss-driven MS3 acquisition: method evaluation through an in vitro study on cytochrome c oxidase modifications.

Publications

Detection and identification of 4-hydroxy-2-nonenal Schiff-base adducts along with products of Michael addition using data-dependent neutral loss-driven MS3 acquisition: method evaluation through an in vitro study on cytochrome c oxidase modifications.

Similar Datasets

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