|Design, synthesis, and application of a hydrazide-functionalized isotope-coded affinity tag for the quantification of oxylipid-protein conjugates.
|Year of Publication
|Han B, Stevens JF, Maier CS
|2007 May 01
|Acrolein, Aldehydes, Alkenes, Animals, Carbon Isotopes, Chromatography, High Pressure Liquid, Hydrazines, Lipid Peroxidation, Lipids, Mitochondria, Heart, Mitochondrial Proteins, Molecular Probes, Molecular Structure, Rats, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
An isotopically coded affinity probe was developed and evaluated for the characterization and quantification of proteins adducted by 2-alkenals derived from lipid peroxidation (LPO) processes. Lipid-derived 2-alkenals, such as acrolein and 4-hydroxy-2-nonenal (HNE), have the ability to react with cysteine, histidine, and lysine residues in proteins, thus causing protein damage and loss of protein function. Such modifications of proteins are difficult to characterize in biological samples by mass spectrometry due to the complexity of protein extracts and the low abundance of adducted proteins. The novel aldehyde-reactive, hydrazide-functionalized, isotope-coded affinity tag (HICAT) described in this study was found effective for the selective isolation, detection, and quantification of Michael-type adducts of 2-alkenals with proteins using a combination of affinity isolation, nanoLC, and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS). The chemical and mass spectrometric properties of the new probe are demonstrated on a model protein treated with HNE. The efficacy of HICAT for the analysis of complex samples was tested using preparations of mitochondrial proteins that were modified in vitro with HNE. The potential of the HICAT strategy for the identification, characterization, and quantification of in vivo oxylipid-protein conjugates is demonstrated on cardiac mitochondrial protein preparations, in which, for example, the ADP/ATP translocase 1 was found adducted to the 2-alkenals, acrolein and 4-hydroxy-2-hexenal, at Cys-256.
|AG025372 / AG / NIA NIH HHS / United States
ES00210 / ES / NIEHS NIH HHS / United States