旧探针的新作用：通过N'-氨氧基甲基羰基肼基d-生物素亲和标记脂质蛋白偶联物。,Analytical Chemistry

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旧探针的新作用：通过N'-氨氧基甲基羰基肼基d-生物素亲和标记脂质蛋白偶联物。
Analytical Chemistry ( IF 6.7 ) Pub Date : 2006 Oct 1 , DOI: 10.1021/ac0607257
Juan Chavez ₁ , Jianyong Wu ₁ , Bingnan Han ₁ , Woon-Gye Chung ₁ , Claudia S. Maier ₁

Affiliation

自由基，亲电试剂和内源性反应中间体是在正常的生理过程中产生的，能够修饰DNA，脂质和蛋白质。但是，由反应性物种引起的蛋白质氧化修饰水平的升高与氧化应激介导的疾病，神经变性和衰老的病因和病理学有关。据报道，基于质谱的方法有助于羰基修饰蛋白的鉴定和表征。该方法使用N'-氨氧基甲基羰基肼基d-生物素，一种生物素化的羟胺衍生物，与氧化修饰的蛋白质中的醛/酮基形成肟衍生物。在本文中，该方法针对一类经羰基修饰的蛋白质进行了验证，即脂质肽和蛋白质共轭物，是由具有亲核肽侧链的α，β-不饱和醛脂质过氧化产物的迈克尔加成型共轭反应形成的。“旧”探针的这种新应用已用于检测DNA链中的无碱基位点，将生物素部分引入了脂质肽结合物中。然后使用抗生物素蛋白亲和捕获使生物素修饰的脂质肽缀合物富集。所描述的方法代表了用于氧化肽和蛋白质的肼基衍生化方法的一种有吸引力的替代方法，因为可以省去bond键转化为化学上更稳定的肼键所必需的还原步骤。标记的脂质肽偶联物的串联质谱表明，在碰撞诱导的解离实验中，生物素部分至少部分保留在碎片离子上，这是使用自动数据库搜索未解释的串联质谱的先决条件。概述了所报道的方法用于脂质肽缀合物的检测，鉴定和表征，但是标记化学方法也可能适用于其他羰基修饰的蛋白质。

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New role for an old probe: affinity labeling of oxylipid protein conjugates by N'-aminooxymethylcarbonylhydrazino d-biotin.

Free radicals, electrophiles, and endogenous reactive intermediates are generated during normal physiological processes and are capable of modifying DNA, lipids, and proteins. However, elevated levels of oxidative modifications of proteins by reactive species are implicated in the etiology and pathology of oxidative stress-mediated diseases, neurodegeneration, and aging. A mass spectrometry-based approach is reported that aids to the identification and characterization of carbonyl-modified proteins. The method uses N'-aminooxymethylcarbonylhydrazino d-biotin, a biotinylated hydroxylamine derivative that forms an oxime derivative with the aldehyde/keto group found in oxidatively modified proteins. In this paper, the method is demonstrated for one class of carbonyl-modified proteins, namely, oxylipid peptide and protein conjugates formed by Michael addition-type conjugation reactions of alpha,beta-unsaturated aldehydic lipid peroxidation products with nucleophilic peptide side chains. This new application of an "old" probe, which has been used for the detection of abasic sites in DNA strands, introduces a biotin moiety into the oxylipid peptide conjugate. The biotin-modified oxylipid peptide conjugate is then amenable to enrichment using avidin affinity capture. The described method represents an attractive alternative to hydrazine-based derivatization methods for oxidized peptides and proteins because the reduction step necessary for the transformation of the hydrazone bond to the chemically more stable hydrazine bond can be omitted. Tandem mass spectrometry of the labeled oxylipid peptide conjugates indicates that the biotin moiety is at least partially retained on the fragment ion during the collisionally induced dissociation experiments, a prerequisite for the use of automated database searching of uninterpreted tandem mass spectra. The reported approach is outlined for the detection, identification, and characterization of oxylipid peptide conjugates, but the labeling chemistry may also be applicable to other carbonyl-modified proteins.

更新日期：2017-01-31

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