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

Abstract

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.

Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria

“…SPH chemistry of peptide carbonyls [35,36] permitted the removal of non-modified species and, thereby, facilitated the identification of HNE-modified peptides in the proteome. We have previously shown that this enrichment technique significantly improved the quality of MS/MS spectra by increasing the number of ion counts and reducing interference during precursor-ion isolation and fragmentation in brain derived mitochondria [23,36]. We, again, utilized this enrichment method in this study to “fish-out” peptide carbonyls from the tryptic digest of HNE-treated mitochondria protein fraction of the rat liver for modification-directed identification.…”

“…Due to its chemical nature, HNE easily undergoes Michael addition on the side chain functional group of Cys, His, and Lys, respectively [19]. It can also produce Schiff bases with the ε-amino group of Lys, but the corresponding kinetics are inherently slow and Schiff-base formation is also reversible [20,21] making Michael-adducts of HNE the predominant [21–23] and most reliable products for exploring protein carbonylation through proteomics [21,23,24]. …”

Angiotensin II modification by decomposition products of linoleic acid-derived lipid hydroperoxide

“…In most cases, the specific modification sites were identified and the consequent biological effects were elucidated. ONE-and HNE-modified proteins have also been found in human red blood cells [36], liver fractions from a rat model of chronic alcoholic disease [37], rat brain tissues [38], and adipose tissues from obese insulin-resistant mice [39]. However, the identification of adduction sites was often challenging because of complex biological matrices and required in vitro experiment using recombinant proteins.…”

Development of an automated on-line pepsin digestion–liquid chromatography–tandem mass spectrometry configuration for the rapid analysis of protein adducts of chemical warfare agents

“…For example, a nerve agent exposure will result in the formation of adducts to human butyryl cholinesterase (HuBuChE) [5], whereas the vesicant sulfur mustard will give rise to rather random adducts, e.g., to human serum albumin (HSA) [6][7][8]. Similar covalent protein adducts are often observed in drug toxicology (see, e.g., [9][10][11]), in metabolism (e.g., [12][13][14][15][16]) and from post-translational modification (e.g., [17,18]). The analysis of covalent protein adducts is usually performed by enzymatic digestion of the modified protein and subsequent mass spectrometric analysis of the resultant peptide or amino acid adducts.…”