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 - Apr 2010

Keywords

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

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