Isotope-coded dimethyl tagging for differential quantification of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation

Navin Rauniyar, Laszlo Prokai

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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 12CHO) and heavy (D 13CDO) 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.

Original languageEnglish
Pages (from-to)976-985
Number of pages10
JournalJournal of Mass Spectrometry
Volume46
Issue number10
DOIs
StatePublished - 1 Oct 2011

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Carbonylation
Isotopes
Lipids
Peptides
Proteins
Methylation
Oxidative stress
Liquid chromatography
Proteome
Membrane Lipids
Unsaturated Fatty Acids
Aldehydes
Washing
Formaldehyde
Lysine
Amines
Mass spectrometry
4-hydroxy-2-nonenal
Labels
Blood Proteins

Keywords

  • 4-hydroxy-2-nonenal
  • LC-MS/MS
  • differential quantitative proteomics
  • protein carbonylation
  • solid-phase hydrazide chemistry
  • stable-isotope tagging

Cite this

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title = "Isotope-coded dimethyl tagging for differential quantification of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation",
abstract = "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 12CHO) and heavy (D 13CDO) 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.",
keywords = "4-hydroxy-2-nonenal, LC-MS/MS, differential quantitative proteomics, protein carbonylation, solid-phase hydrazide chemistry, stable-isotope tagging",
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T1 - Isotope-coded dimethyl tagging for differential quantification of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation

AU - Rauniyar, Navin

AU - Prokai, Laszlo

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N2 - 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 12CHO) and heavy (D 13CDO) 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.

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