Design, Synthesis, and Application of a Hydrazide-Functionalized Isotope-Coded Affinity Tag for the Quantification of Oxylipid-Protein Conjugates

详细信息    查看全文

• 作者：Bingnan Han ; Jan F. Stevens ; Claudia S. Maier
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：May 1, 2007
• 年：2007
• 卷：79
• 期：9
• 页码：3342 - 3354
• 全文大小：487K
• 年卷期：v.79,no.9(May 1, 2007)
• ISSN：1520-6882

文摘

An isotopically coded affinity probe was developed andevaluated for the characterization and quantification ofproteins adducted by 2-alkenals derived from lipid peroxidation (LPO) processes. Lipid-derived 2-alkenals, suchas acrolein and 4-hydroxy-2-nonenal (HNE), have theability to react with cysteine, histidine, and lysine residuesin proteins, thus causing protein damage and loss ofprotein function. Such modifications of proteins aredifficult to characterize in biological samples by massspectrometry due to the complexity of protein extracts andthe low abundance of adducted proteins. The novelaldehyde-reactive, hydrazide-functionalized, isotope-codedaffinity tag (HICAT) described in this study was foundeffective for the selective isolation, detection, and quantification of Michael-type adducts of 2-alkenals withproteins using a combination of affinity isolation, nanoLC,and matrix-assisted laser desorption ionization tandemmass spectrometry (MALDI-MS/MS). The chemical andmass spectrometric properties of the new probe aredemonstrated on a model protein treated with HNE. Theefficacy of HICAT for the analysis of complex samples wastested using preparations of mitochondrial proteins thatwere modified in vitro with HNE. The potential of theHICAT strategy for the identification, characterization, andquantification of in vivo oxylipid-protein conjugates isdemonstrated on cardiac mitochondrial protein preparations, in which, for example, the ADP/ATP translocase 1was found adducted to the 2-alkenals, acrolein and4-hydroxy-2-hexenal, at Cys-256.