Combined Proteomic Approaches for the Identification of Specific Amino Acid Residues Modified by 4-Hydroxy-2-Nonenal under Physiological Conditions
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- 作者：Daro Mndez ; Maria Luisa Hernez ; Amalia Diez ; Antonio Puyet ; Jos M. Bautista
- 刊名：Journal of Proteome Research
- 出版年：2010
- 出版时间：November 5, 2010
- 年：2010
- 卷：9
- 期：11
- 页码：5770-5781
- 全文大小：378K
- 年卷期：v.9,no.11(November 5, 2010)
- ISSN：1535-3907
文摘
Proteins modified by 4-hydroxy-2-nonenal (HNE) are cellular markers of oxidative stress in health and disease. HNE is generated by free radical chain reactions during oxidative stress as a major end-product of the oxidative fatty acid metabolism. Identification and quantitative analysis of HNE-modified proteins are readily performed by using specific antibodies raised against them. Further on, the identification of the amino acid residues involved in the HNE-modification is an additional step in proteomic post-transcriptional modification analysis to explain the nature of the specificity underlying oxidative stress mechanisms. For this purpose, a combined protocol of immune-detection, peptide enrichment, mass spectrometry, and de novo protein sequencing has been developed. The methodology was first examined in the model protein bovine serum albumin (BSA), allowing the comparison of matrix-assisted laser desorption/ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) performance and sensitivity. Peptide enrichment was optimized by affinity chromatography on HNE-BSA resulting in increased sensitivity. Identification of amino acid residues modified by HNE was finally ascertained by de novo sequencing analysis. The improved methodology was demonstrated on human erythrocyte membrane proteins allowing the identification of HNE-lysine and HNE-histidine Michael adducts in the β-spectrin under physiological conditions.