title: Proteomic approaches to identifying carbonylated proteins in brain tissue.
abstract: Oxidative stress plays a critical role in the pathogenesis of a number of diseases. The carbonyl end products of protein oxidation are among the most commonly measured markers of oxidation in biological samples. Protein carbonyl functional groups may be derivatized with 2,4-dinitrophenylhydrazine (DNPH) to render a stable 2,4-dinitrophenylhydrazone-protein (DNP-protein) and the carbonyl contents of individual proteins then determined by two-dimensional electrophoresis followed by immunoblotting using specific anti-DNP antibodies. Unfortunately, derivatization of proteins with DNPH could affect their mass spectrometry (MS) identification. This problem can be overcome using nontreated samples for protein identification. Nevertheless, derivatization could also affect their mobility, which might be solved by performing the derivatization step after the initial electrophoresis. Here, we compare two-dimensional redox proteome maps of mouse cerebellum acquired by performing the DNPH derivatization step before or after electrophoresis and detect differences in protein patterns. When the same approach is used for protein detection and identification, both methods were found to be useful to identify carbonylated proteins. However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH reaction was performed after transblotting. Comparative data on protein identification using both methods are provided.
authors.affiliation: Department of Biochemistry and Molecular Biology IV, Facultad de Veterinaria, Instituto de Investigación Hospital 12 de Octubre, Universidad Complutense de Madrid, Ciudad Universitaria, 28040 Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Linares
authors.forename: Patricia
authors.initials: P
authors.lastname: Marín-Garcíía
authors.forename: Darío
authors.initials: D
authors.lastname: Méndez
authors.forename: Antonio
authors.initials: A
authors.lastname: Puyet
authors.forename: Amalia
authors.initials: A
authors.lastname: Diez
authors.forename: José M
authors.initials: JM
authors.lastname: Bautista
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D051379:Mice; D015394:Molecular Structure; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D018384:Oxidative Stress; D010659:Phenylhydrazines / Q000737:chemistry; D050050:Protein Carbonylation; D040901:Proteomics / Q000379:methods*; D017382:Reactive Oxygen Species / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D009419:Nerve Tissue Proteins; D010659:Phenylhydrazines; D017382:Reactive Oxygen Species; C004787:2,4-dinitrophenylhydrazine