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

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 mM hydrogen peroxide to induce oxidative stress. Biotinylation of yeast protein was accomplished during extraction at 4 degrees C 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.

Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH

“…In particular, all of the glycolytic and cytosolic metabolic enzymes listed in Table 3, with the exception of Lys9p, were identified in both types of studies [24,46,47]. In other functional categories, Sod1p and cyclophilin Cpr1p were similarly identified as carbonylated in the Δidp2Δzwf1 mutant shifted to oleate medium, and in wild-type glucose-grown hydrogen peroxide-challenged cells [24].…”

“…(These effects were much less pronounced with a shift of the mutant strain to acetate medium.) We used two-dimensional electrophoresis to examine carbonylated proteins in the mutant strain shifted to oleate in the absence or presence of glutathione and DTT, with the goal of comparing results with our data on disulfide bond-containing proteins, as well as with results of several reported studies of carbonylation in yeast cells grown on glucose with exogenous oxidants [24,46,47].…”

Oxidation of proteins: Basic principles and perspectives for blood proteomics

“…The same chemistry can be used in combination with gel electrophoresis, followed by an immunodetection [88,89]. Alternatively, Yoo and Regnier [90] have developed a biotinylation strategy for the specific labelling of carbonylated proteins after 2-DE.…”

Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation

“…It should be mentioned that enolase-α has been demonstrated to be an early and specific target for oxidative damage by carbonylation in different cell systems, ranging from yeast [52,53] to humans [54,55]. However, the fact that carbonylation of enolase-α appears not to alter its pI [55] suggests that this process may not be directly responsible for the observed charge shifts for enolase-α in the 0 Gy (95%) & 5 Gy (5%), and 5 Gy (100%) samples.…”