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• W2079349780 abstract "Abstract A method for detecting carbonylated proteins in two‐dimensional electrophoresis (2‐DE) was developed using biotinylation and avidin‐fluorescein isothiocyanate (FITC) affinity staining. The method was used to examine oxidatively modified proteins associated with oxidative stress. Carbonyl formation in proteins was first examined in a model system by subjecting bovine serum albumin (BSA) and ribonuclease A (RNase A) to metal‐catalyzed oxidation (MCO). Carbonyl group formation was found to occur at multiple sites along with a small amount of polypeptide chain cleavage. In vivo studies were conducted in yeast cell cultures using 5 m M hydrogen peroxide to induce oxidative stress. Biotinylation of yeast protein was accomplished during extraction at 4°C in a lysis buffer containing 5 m M biotin‐hydrazide. Biotin‐hydrazide forms a Schiff base with a carbonyl group on an oxidized protein that is subsequently reduced before electrophoresis. Proteins were separated by either 2‐DE or sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Biotinylated species were detected using avidin‐FITC affinity staining. Detection sensitivity with biotinylated proteins was five times higher than achieved by silver staining. The limit of detection with avidin‐FITC staining approached 0.64 pmol of protein‐associated carbonyls. Twenty carbonylated proteins were identified in the proteome of yeast following oxidative stress with hydrogen peroxide. Matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) analysis of tryptic peptides was used to identify peptides extracted from gels. Aconitase, heat shock protein SSA1 and SSC1, pyruvate decarboxylase isozyme 1, pyruvate kinase 1, enolase 1 and 2, phosphoglycerate kinase, fructose‐bisphosphate aldorase, and glyceraldehyde‐3‐phosphate dehydrogenase were among the major targets of oxidative stress." @default.
• W2079349780 created "2016-06-24" @default.
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• W2079349780 date "2004-05-01" @default.
• W2079349780 modified "2023-10-10" @default.
• W2079349780 title "Proteomic analysis of carbonylated proteins in two-dimensional gel electrophoresis using avidin-fluorescein affinity staining" @default.
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• W2079349780 doi "https://doi.org/10.1002/elps.200405890" @default.
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