title: Fluorescence labeling of carbonylated lipids and proteins in cells using coumarin-hydrazide.
abstract: Carbonylation is a generic term which refers to reactive carbonyl groups present in biomolecules due to oxidative reactions induced by reactive oxygen species. Carbonylated proteins, lipids and nucleic acids have been intensively studied and often associated with onset or progression of oxidative stress related disorders. In order to reveal underlying carbonylation pathways and biological relevance, it is crucial to study their intracellular formation and spatial distribution. Carbonylated species are usually identified and quantified in cell lysates and body fluids after derivatization using specific chemical probes. However, spatial cellular and tissue distribution have been less often investigated. Here, we report 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 specificity of coumarin-hydrazide was confirmed in time- and dose-dependent experiments using human primary fibroblasts stressed with paraquat and compared with conventional DNPH-based immunocytochemistry. Both techniques stained carbonylated species accumulated in cytoplasm with strong perinuclear clustering. Using a complimentary array of analytical methods specificity of coumarin-hydrazide probe towards both protein- and lipid-bound carbonyls has been shown. Additionally, co-distribution of carbonylated species and oxidized phospholipids was demonstrated.
authors.affiliation: Faculty of Chemistry and Mineralogy, Institute of Bioanalytical Chemistry, Leipzig, Germany; Center for Biotechnology and Biomedicine, Universität Leipzig, Leipzig, Germany.
authors.forename: Venukumar
authors.initials: V
authors.lastname: Vemula
authors.affiliation: Faculty of Chemistry and Mineralogy, Institute of Bioanalytical Chemistry, Leipzig, Germany; Center for Biotechnology and Biomedicine, Universität Leipzig, Leipzig, Germany.
authors.forename: Zhixu
authors.initials: Z
authors.lastname: Ni
authors.affiliation: Faculty of Chemistry and Mineralogy, Institute of Bioanalytical Chemistry, Leipzig, Germany; Center for Biotechnology and Biomedicine, Universität Leipzig, Leipzig, Germany. Electronic address: maria.fedorova@bbz.uni-leipzig.de.
authors.forename: Maria
authors.initials: M
authors.lastname: Fedorova
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D003374:Coumarins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006834:Hydrazines / Q000737:chemistry*; D007150:Immunohistochemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress / Q000187:drug effects; D010269:Paraquat / Q000633:toxicity; D010743:Phospholipids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D050050:Protein Carbonylation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D053719:Tandem Mass Spectrometry
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D006834:Hydrazines; D010743:Phospholipids; D011506:Proteins; C446799:dinitrophenylhydrazine; C030123:coumarin; D010269:Paraquat
keywords: Coumarin-hydrazide; Dinitrophenyl hydrazine; Fluorescence microscopy; Protein and lipid carbonylation; Spatial distribution