Proteomic Mapping of 4-Hydroxynonenal Protein Modification Sites by Solid-Phase Hydrazide Chemistry and Mass Spectrometry

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• 作者：Mikel R. Roe ; Hongwei Xie ; Sricharan Bandhakavi ; Timothy J. Griffin
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：May 15, 2007
• 年：2007
• 卷：79
• 期：10
• 页码：3747 - 3756
• 全文大小：243K
• 年卷期：v.79,no.10(May 15, 2007)
• ISSN：1520-6882

文摘

The modification of proteins by the cytotoxic, reactivealdehyde 4-hydroxynonenal (HNE) is known to alterprotein function and impair cellular mechanisms. In orderto identify susceptible amino acid sites of HNE modification within complex biological mixtures by microcapillaryliquid chromatography and linear ion trap tandem massspectrometry, we have developed a solid-phase captureand release strategy that utilizes reversible hydrazidechemistry to enrich HNE-modified peptides. To maximizethe detection of fragment ions diagnostic of HNE modification, both neutral loss-dependent acquisition of MS/MS/MS spectra and the pulsed Q dissociation operationmode were employed. When the solid-phase hydrazideenrichment strategy was applied to a yeast lysate treatedwith HNE, 125 distinct amino acid sites of HNE modification were mapped on 67 different proteins. The endogenous susceptibility of many of these proteins to HNEmodification was demonstrated by analyzing HNE-treatedyeast cell cultures with a complementary biotin hydrazideenrichment strategy. Further analysis revealed that themajority of amino acid sites susceptible to HNE modification were histidine residues, with most of these sites beingflanked by basic amino acid residues, and predicted tobe solvent exposed. These results demonstrate the effectiveness of this novel strategy as a general platform forproteome-scale identification of amino acid sites susceptible to HNE modification from within complex mixtures.