PP16 - Fluorescent microscopy study of intracellular distribution of carbonylated proteins and lipids

Abstract

Carbonylation is an important oxidative modification of biomolecules and widely accepted as a biomarker of oxidative stress (OS). Carbonylated proteins, lipids, and nucleic acids have been intensively studied and were often linked to the onset or progression of OS related disorders. Carbonylated species are usually identified and quantified in cell lysates and body fluids after derivatization with specific chemical probes. Several analytical methods for identification and quantification of carbonylated biomolecules have been reported. However, in order to understand cellular carbonylation pathways and reveal their biological relevance it is crucial to study their intracellular formation and spatial distribution. Here, we used coumarin-hydrazide, a fluorescent chemical probe, for time- and cost-efficient labeling of cellular carbonyls followed by fluorescence microscopy to evaluate their intracellular formation both in time and space. The protocol was verified in a cellular model of paraquat induced OS and compared with a conventional DNPH-based immunocytochemistry. The specificity of coumarin-hydrazide towards carbonylated proteins and lipids was confirmed by a wide range of analytical techniques including gel electrophoresis, thin layer chromatography, and mass spectrometry. Additionally, co-distribution with oxidized lipids was evaluated by confocal microscopy using oxidized phosphatidylcholine specific natural antibodies. We have applied this method to detect carbonyls in several cellular models of oxidative stress including paracetamol induced hepatocyte toxicity and nitrosative stress in primary cardiomyocytes. A strong increase in biomolecule oxidation was observed in all studied OS models and most of the carbonylated species were accumulated in perinuclear space. Using confocal microscopy, co-localization of carbonylated biomolecules with endoplasmic reticulum was also demonstrated.