Tandem mass spectrometric analysis of protein and peptide adducts of lipid peroxidation-derived aldehydes Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/pc289m14c

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  • The adduction of proteins and other biomolecules by electrophilic lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), malondialdehyde (MDA) or acrolein (ACR) is thought to be an initiating and/or propagating factor in the pathophysiology of several diseases such as atherosclerosis, diabetes, Alzheimer's, Parkinson's and other age-related disorders. The identification of protein sites modified by oxylipids is of key relevance for advancing our understanding how oxidative damage affects structure and function of proteins. Here, the use of MALDI tandem mass spectrometry with high energy collision-induced dissociation (CID) on a TOF/TOF instrument for sequencing oxylipid-peptide conjugates was systematically studied. Three synthesized model peptides containing one nucleophilic residue (i.e. Cys, His or Lys) were reacted with MDA, HNE, ONE and ACR. MALDI-MS analysis and MS/MS analysis were performed to confirm the adduct type and the modification sites. Michael adducts and Schiff bases were the predominant products under pH 7.4 within 2 hours. All MS/MS spectra of Michael adducts show the neutral loss of the oxylipid moiety ions. MS/MS spectra of Cys-containing peptide oxylipid conjugates exhibit additional characteristic neutral loss of HS-oxylipid moiety ions. MS/MS spectra of His-containing peptide oxylipid conjugates show characteristic oxylipid-containing His immonium ions. Spectra of Lys-containing peptide oxylipid conjugates (Schiff base) also show oxylipid-containing Lys immonium ions. However, there is no neutral loss of the oxylipid moiety ion for these Schiff bases. Determining the extent or relative amounts of the oxidative damage in cells could provide valuable insights into the molecular mechanisms of the diseases caused by oxidative stress. Relative quantitation of oxylipid-modified proteins in biological samples is a challenging problem because of the complexity and extreme dynamic range that characterize these samples. In this study, the reagents, N'-aminooxymethylcarbonylhydrazino-D-biotin (ARP) and iodoacetyl-PEO2- biotin (IPB), were used to enrich acrolein-modified Cys-containing peptides and the corresponding unmodified ones from subsarcolemmal mitochondria (SSM). The ratios between them were determined by nanoLC-SRM analysis. Model Cys-containing peptides labeled with ARP-acrolein and IPB were employed to demonstrate this method. Seven acrolein-modifed Cys-containing peptides from five mitochondrial proteins were quantified. The ratios for those seven peptides from the CCl₄-treated rats are higher than the control ones indicating that the ratios of acrolein-modified peptides to unmodified ones are potential markers of oxidative stress in vivo. Age-dependent changes of protein carbonyls were investigated in subsarcolemmal mitochondria by using LC-SRM analysis of distinct ACR-modified Cys-containing peptides. Immunochemical analysis using an anti-ACR monoclonal antibody supported an increase of proteins modified by acrolein with age. However, total protein carbonyls measurement using ARP in Western blot analysis did not conform to this change suggesting that age-related changes in protein carbonyls are complex and would benefit from more specific measurement protocols.
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