Rapid characterization of covalent modifications to rat brain mitochondrial proteins after ex vivo exposure to 4-hydroxy-2-nonenal by liquid chromatography-tandem mass spectrometry using data-dependent and neutral loss-driven MS3 acquisition.
The modification of mitochondrial proteins enriched from rat forebrain by the major lipid peroxidation product 4-hydroxy-2-nonenal (HNE) was investigated using high performance liquid chromatography (HPLC) and tandem mass spectrometry. Subcellular fractionation in conjunction with a 'shotgun-based' approach that involved both conventional data-dependent and neutral loss (NL)-driven MS(3) data acquisition on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT) was utilized. Using a relatively rapid linear HPLC gradient (1 h) for complex mixture analysis, 24 sites of HNE modification on 15 unique proteins were identified which corresponded exclusively to Michael adduct formation on histidine residues. Since a number of HNE-modified peptides produced a predominant HNE NL fragment-ion signal upon collision-induced dissociation (CID), NL-driven MS(3) data-dependent acquisition was a valuable method to enhance fragmentation information for these particular modified peptides. Of the 24 HNE modification sites identified, approximately 25% were determined from the MS(3) spectra alone. We envision the reported methodology as an efficient screening approach for HNE modification site selectivity that could ultimately provide a foundation for the development of targeted schemes for the characterization of in vivo HNE-protein adducts.
Stevens
Stanley M
SM
Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699, USA.
Rauniyar
Navin
N
Prokai
Laszlo
L
D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001923:Brain Chemistry / Q000187:drug effects*; D002851:Chromatography, High Pressure Liquid; D017357:Cyclotrons; D003627:Data Interpretation, Statistical; D030562:Databases, Protein; D005583:Fourier Analysis; D007202:Indicators and Reagents; D008297:Male; D024101:Mitochondrial Proteins / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D013482:Superoxide Dismutase / Q000737:chemistry; D000072105:Superoxide Dismutase-1; D053719:Tandem Mass Spectrometry
D000447:Aldehydes; D007202:Indicators and Reagents; D024101:Mitochondrial Proteins; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D010447:Peptide Hydrolases; C027576:4-hydroxy-2-nonenal