Proteomic analysis of carbonylated proteins in two-dimensional gel electrophoresis using avidin-fluorescein affinity staining

被引:95
作者
Yoo, YS [1 ]
Regnier, FE [1 ]
机构
[1] Purdue Univ, Dept Chem, W Lafayette, IN 47907 USA
关键词
avidin-fluorescein isothiocyanate; biotin-hydrazide; matrix-assisted laser desorption/ionization; time of flight mass spectrometry; metal-catalyzed oxidation; peptide mass fingerprinting; protein carbonyls; two-dimensional gel electrophoresis;
D O I
10.1002/elps.200405890
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 mm hydrogen peroxide to induce oxidative stress. Biotinylation of yeast protein was accomplished during extraction at 4degreesC in a lysis buffer containing 5 mm 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.
引用
收藏
页码:1334 / 1341
页数:8
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