title: Analysis of oxidative modification of proteins.
abstract: Proteins are targets of oxidative modification. This unit describes detailed procedures for the analysis of popular indices of protein oxidation including protein carbonyl formation, loss of protein thiols, and nitrotyrosine and dityrosine formation, as well as isoaspartate formation. Procedures are detailed for the analysis of protein carbonyls labeled with 2,4-dinitrophenylhydrazine, tritiated sodium borohydride, and biotin-hydrazide, followed by detection measurements that are based on the distinguishing feature of each labeling chemical. Methods are outlined for the determination of protein cysteine oxidation by quantifying the loss of free protein thiols using radiolabeled [(14)C]-iodoacetamide. Protocols are described for the measurement of protein dityrosine by gas chromatography/mass spectrometry, as are the details for the detection of protein nitrotyrosine by a competitive ELISA approach. Finally, methods are described for the quantification of protein-bound isoaspartate using protein-L-isoaspartyl methyltransferase that converts aberrant L-isoaspartyl residues in peptides and proteins to normal aspartyl residues.
authors.affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas, USA.
authors.forename: Liang-Jun
authors.initials: LJ
authors.lastname: Yan
mesh_terms: D001671:Biochemistry / Q000379:methods*; D001710:Biotin / Q000031:analogs & derivatives / Q000032:analysis; D001894:Borohydrides / Q000032:analysis; D003545:Cysteine / Q000378:metabolism; D026581:Isoaspartic Acid / Q000032:analysis; D007553:Isotope Labeling; D010084:Oxidation-Reduction; D010659:Phenylhydrazines / Q000032:analysis; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000032:analysis; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
chemical_list: D001894:Borohydrides; D026581:Isoaspartic Acid; D010659:Phenylhydrazines; D011506:Proteins; D013438:Sulfhydryl Compounds; C004787:2,4-dinitrophenylhydrazine; D014443:Tyrosine; C053311:biotin hydrazide; D001710:Biotin; C025364:sodium borohydride; C007543:dityrosine; D003545:Cysteine