Identification of carbonylation sites in apomyoglobin after exposure to 4-hydroxy-2-nonenal by solid-phase enrichment and liquid chromatography-electrospray ionization tandem mass spectrometry.
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 a model 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 ECD was employed. We have concluded that solid-phase enrichment with 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 via Michael addition to its histidine residues.
Rauniyar
Navin
N
Department of Molecular Biology & Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107-2699, USA.
Prokai-Tatrai
Katalin
K
Prokai
Laszlo
L
D000447:Aldehydes / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D006736:Horses; D018482:Muscle, Skeletal / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D050050:Protein Carbonylation; D052616:Solid Phase Extraction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D000447:Aldehydes; D001059:Apoproteins; D009211:Myoglobin; D010446:Peptide Fragments; C010857:apomyoglobin; D014357:Trypsin; C027576:4-hydroxy-2-nonenal