title: Mass spectrometry-based proteomics of oxidative stress: Identification of 4-hydroxy-2-nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins.
abstract: Modification of proteins by 4-hydroxy-2-nonenal (HNE), a reactive by-product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age-related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff-base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff-base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS "signatures" of HNE-modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE-modified lysozyme into an electrospray quadrupole time-of-flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC-MS/MS, we found that, in addition to N-terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.
authors.affiliation: Biochemistry and Proteomics Group, Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY, USA.
authors.forename: Roshanak
authors.initials: R
authors.lastname: Aslebagh
authors.affiliation: Research Service, VA Western NY Healthcare System, Buffalo, NY, USA.|Departments of Ophthalmology and Biochemistry, SUNY- University at Buffalo, Buffalo, NY, USA.|SUNY Eye Institute, Buffalo, NY, USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Pfeffer
authors.affiliation: Research Service, VA Western NY Healthcare System, Buffalo, NY, USA.|Departments of Ophthalmology and Biochemistry, SUNY- University at Buffalo, Buffalo, NY, USA.|SUNY Eye Institute, Buffalo, NY, USA.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Fliesler
authors.affiliation: Biochemistry and Proteomics Group, Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY, USA. cdarie@clarkson.edu.
authors.forename: Costel C
authors.initials: CC
authors.lastname: Darie
mesh_terms: D000447:Aldehydes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D008956:Models, Chemical; D009113:Muramidase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D036103:Nanotechnology / Q000379:methods; D018384:Oxidative Stress; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000447:Aldehydes; D000596:Amino Acids; D012710:Serum Albumin, Bovine; D009113:Muramidase; C027576:4-hydroxy-2-nonenal
keywords: 4-Hydroxy-2-nonenal; Mass spectrometry; Michael addition; Peptide modification; Proteomics; Pyrrole adducts; Schiff-base