Paper presented at the 25th Informal Meeting on Mass Spectrometry, Nyiregyháza-Sóstó, Hungary, 6–10 May, 2007.
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†
Version of Record online: 17 DEC 2007
Copyright © 2007 John Wiley & Sons, Ltd.
Journal of Mass Spectrometry
Volume 42, Issue 12, pages 1599–1605, December 2007
How to Cite
Stevens, S. M., Rauniyar, N. and Prokai, L. (2007), 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. J. Mass Spectrom., 42: 1599–1605. doi: 10.1002/jms.1349
- Issue online: 17 DEC 2007
- Version of Record online: 17 DEC 2007
- Manuscript Accepted: 14 OCT 2007
- Manuscript Received: 17 JUN 2007
- National Institutes of Health; Robert A. Welch Foundation. Grant Number: AG025384
- Fourier transform ion cyclotron resonance mass spectrometry;
- tandem mass spectrometry;
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 MS3 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 MS3 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 MS3 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. Copyright © 2007 John Wiley & Sons, Ltd.