Identification of carbonylation sites in apomyoglobin after exposure to 4-hydroxy-2-nonenal by solid-phase enrichment and liquid chromatography- electrospray ionization tandemmass spectrometry

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Abstract

Identification of protein carbonylation because of covalent attachment of a lipid peroxidation end-product was performed by combining proteolytic digestion followed by solid-phase hydrazide enrichment and liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using both collision-induced dissociation (CID) and electron capture dissociation (ECD). To evaluate this approach, we selected apomyoglobin and 4-hydroxy-2-nonenal (4-HNE) as amodel protein and a representative end-product of lipid peroxidation, respectively. Although the characteristic elimination of 4-HNE (156 Da) in CID was found to serve as a signature tag for the modified peptides, generation of nearly complete fragment ion series because of efficient peptide backbone cleavage (in most cases over 75%) and the capability to retain the labile 4-HNE moiety of the tryptic peptides significantly aided the elucidation of primary structural information and assignment of exact carbonylation sites in the protein,when ECDwas employed.Wehave concluded that solid-phase enrichmentwith both CID-and ECD-MS/MS are advantageous during an in-depth interrogation and unequivocal localization of 4-HNE-induced carbonylation of apomyoglobin that occurs viaMichael addition to its histidine residues.

Original languageEnglish
Pages (from-to)398-410
Number of pages13
JournalJournal of Mass Spectrometry
Volume45
Issue number4
DOIs
StatePublished - 1 Apr 2010

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Carbonylation
Electrospray ionization
Liquid chromatography
Spectrometry
Peptides
Lipids
Proteins
Electrons
Histidine
Mass spectrometry
apomyoglobin
4-hydroxy-2-nonenal
Ions

Keywords

  • 4-hydroxy-2-nonenal
  • Collision-induced dissociation
  • Electron capture dissociation
  • LC-MS/MS
  • Protein carbonylation
  • Solid-phase hydrazide chemistry

Cite this

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title = "Identification of carbonylation sites in apomyoglobin after exposure to 4-hydroxy-2-nonenal by solid-phase enrichment and liquid chromatography- electrospray ionization tandemmass spectrometry",
abstract = "Identification of protein carbonylation because of covalent attachment of a lipid peroxidation end-product was performed by combining proteolytic digestion followed by solid-phase hydrazide enrichment and liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using both collision-induced dissociation (CID) and electron capture dissociation (ECD). To evaluate this approach, we selected apomyoglobin and 4-hydroxy-2-nonenal (4-HNE) as amodel protein and a representative end-product of lipid peroxidation, respectively. Although the characteristic elimination of 4-HNE (156 Da) in CID was found to serve as a signature tag for the modified peptides, generation of nearly complete fragment ion series because of efficient peptide backbone cleavage (in most cases over 75{\%}) and the capability to retain the labile 4-HNE moiety of the tryptic peptides significantly aided the elucidation of primary structural information and assignment of exact carbonylation sites in the protein,when ECDwas employed.Wehave concluded that solid-phase enrichmentwith both CID-and ECD-MS/MS are advantageous during an in-depth interrogation and unequivocal localization of 4-HNE-induced carbonylation of apomyoglobin that occurs viaMichael addition to its histidine residues.",
keywords = "4-hydroxy-2-nonenal, Collision-induced dissociation, Electron capture dissociation, LC-MS/MS, Protein carbonylation, Solid-phase hydrazide chemistry",
author = "Navin Rauniyar and Katalin Prokai-Tatrai and Laszlo Prokai",
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T1 - Identification of carbonylation sites in apomyoglobin after exposure to 4-hydroxy-2-nonenal by solid-phase enrichment and liquid chromatography- electrospray ionization tandemmass spectrometry

AU - Rauniyar, Navin

AU - Prokai-Tatrai, Katalin

AU - Prokai, Laszlo

PY - 2010/4/1

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N2 - Identification of protein carbonylation because of covalent attachment of a lipid peroxidation end-product was performed by combining proteolytic digestion followed by solid-phase hydrazide enrichment and liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using both collision-induced dissociation (CID) and electron capture dissociation (ECD). To evaluate this approach, we selected apomyoglobin and 4-hydroxy-2-nonenal (4-HNE) as amodel protein and a representative end-product of lipid peroxidation, respectively. Although the characteristic elimination of 4-HNE (156 Da) in CID was found to serve as a signature tag for the modified peptides, generation of nearly complete fragment ion series because of efficient peptide backbone cleavage (in most cases over 75%) and the capability to retain the labile 4-HNE moiety of the tryptic peptides significantly aided the elucidation of primary structural information and assignment of exact carbonylation sites in the protein,when ECDwas employed.Wehave concluded that solid-phase enrichmentwith both CID-and ECD-MS/MS are advantageous during an in-depth interrogation and unequivocal localization of 4-HNE-induced carbonylation of apomyoglobin that occurs viaMichael addition to its histidine residues.

AB - Identification of protein carbonylation because of covalent attachment of a lipid peroxidation end-product was performed by combining proteolytic digestion followed by solid-phase hydrazide enrichment and liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using both collision-induced dissociation (CID) and electron capture dissociation (ECD). To evaluate this approach, we selected apomyoglobin and 4-hydroxy-2-nonenal (4-HNE) as amodel protein and a representative end-product of lipid peroxidation, respectively. Although the characteristic elimination of 4-HNE (156 Da) in CID was found to serve as a signature tag for the modified peptides, generation of nearly complete fragment ion series because of efficient peptide backbone cleavage (in most cases over 75%) and the capability to retain the labile 4-HNE moiety of the tryptic peptides significantly aided the elucidation of primary structural information and assignment of exact carbonylation sites in the protein,when ECDwas employed.Wehave concluded that solid-phase enrichmentwith both CID-and ECD-MS/MS are advantageous during an in-depth interrogation and unequivocal localization of 4-HNE-induced carbonylation of apomyoglobin that occurs viaMichael addition to its histidine residues.

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