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Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser‐induced fluorescence detection
Author(s) -
Feng Juan,
Arriaga Edgar A.
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700262
Subject(s) - chemistry , capillary electrophoresis , skeletal muscle , alexa fluor , chromatography , oxidative phosphorylation , mitochondrion , fluorescence , oxidative stress , proteomics , laser induced fluorescence , electrophoresis , biochemistry , biology , gene , physics , quantum mechanics , endocrinology
Carbonyl‐modified proteins are markers of oxidative damage. Here, we report a new method for detecting and quantifying carbonylated proteins by capillary sieving electrophoresis (CSE) with LIF detection (CSE‐LIF). Alexa 488 hydrazide is used for the specific labeling of carbonyls while 3‐(2‐furoyl) quinoline‐2‐carboxaldehyde (FQ) is used for protein labeling. BSA subjected to metal‐catalyzed oxidation is used to optimize the labeling reactions, confirm the separation power of CSE, and characterize the response of the LIF detector. The method is capable of detecting femtomole (fmol) amounts of carbonyls in proteins with molecular masses ranging from 26 to 30 kDa. Using this method, we determined that mitochondrial proteins isolated from skeletal muscle contains 2.1 ± 0.1 (average ± SD; n = 3) nmol carbonyl/mg protein. The methodology described here should be compatible with the analysis of single cells and needle biopsies taken from oxidative stress animal models.