Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser-induced fluorescence detection

Juan Feng, Edgar Arriaga

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

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. BS A 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.

Original languageEnglish (US)
Pages (from-to)475-482
Number of pages8
JournalELECTROPHORESIS
Volume29
Issue number2
DOIs
StatePublished - Jan 2008

Keywords

  • Capillary sieving electrophoresis
  • Carbonyl labeling
  • LIF
  • Mitochondria
  • Protein labeling

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