title: High-resolution mass spectrometry analysis of protein oxidations and resultant loss of function.
abstract: MS, with or without pre-analysis peptide fractionation, can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen or electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighbouring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose-response inactivation by 4HNE (4-hydroxynonenal) of hBAT (human bile acid CoA:amino acid N-acyltransferase) and CKBB (cytosolic brain isoform of creatine kinase) is associated with site-specific modifications. FT-ICR (Fourier-transform ion cyclotron resonance)-MS using nanoLC (nano-liquid chromatography)-ESI (electrospray ionization)-MS or direct-infusion ESI-MS with gas-phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA. sbarnes@uab.edu
authors.forename: Stephen
authors.initials: S
authors.lastname: Barnes
authors.forename: Erin M
authors.initials: EM
authors.lastname: Shonsey
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Eliuk
authors.forename: David
authors.initials: D
authors.lastname: Stella
authors.forename: Kerri
authors.initials: K
authors.lastname: Barrett
authors.forename: Om P
authors.initials: OP
authors.lastname: Srivastava
authors.forename: Helen
authors.initials: H
authors.lastname: Kim
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Renfrow
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000447:Aldehydes / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D052276:Creatine Kinase, BB Form / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017550:Spectroscopy, Fourier Transform Infrared; D038203:alpha-Crystallin B Chain / Q000235:genetics / Q000378:metabolism
chemical_list: D000447:Aldehydes; C059215:CRYAB protein, human; D015853:Cysteine Proteinase Inhibitors; D038203:alpha-Crystallin B Chain; D000217:Acyltransferases; C026953:bile acid-CoA amino acid N-acyltransferase; D052276:Creatine Kinase, BB Form; C027576:4-hydroxy-2-nonenal
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