Method to site-specificafly identify and quantitate carbonyl end products of protein oxidation using oxidation-dependent element coded affinity tags (O-ECAT) and nanoliquid chromatography Fourier transform mass spectrometry
Method to site-specificafly identify and quantitate carbonyl end products of protein oxidation using oxidation-dependent element coded affinity tags (O-ECAT) and nanoliquid chromatography Fourier transform mass spectrometry
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DOI：
10.1021/pr050299q
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发表时间：
2006-03-01
影响因子：
4.4
通讯作者：
Meares, CF
中科院分区：
文献类型：
--
作者：
Lee, S;Young, NL;Meares, CF
蛋白质氧化与细胞压力、衰老和疾病有关。导致反应性物质的蛋白质氧化引起了特别的兴趣，因为这些反应性氧化产物可能以无介导和不可逆的方式与其他蛋白质或生物分子发生反应，为各种疾病机制提供了潜在的标记物。我们已经开发了一种新的系统来识别和定量，相对于其他状态，在一个给定的蛋白质上的氧化位点。该方法结合了现有方法的优点，增加了多重、定量和探测更多修饰氨基酸的能力，比现有方法有了显著的进步。一种特殊设计的氧化依赖性羰基特异性元素编码亲和质量标签（O-ECAT）， AOD, (（S)-2-（4-（2-氨基）-乙酰氨基）-苄基）-1,4,7,10-四氮杂环十二烷-N，N‘, ‘， N’ ’ -四乙酸，用于共价标记蛋白质氧化成醛或酮的最终产物的残基。O-ECAT可以装载各种金属，从而产生质量对和多重样品的能力。O-ECAT部分也用作句柄。用于鉴定、定量和亲和纯化。蛋白水解后，AOD标记的肽被亲和纯化，并通过纳米液相色谱-傅里叶变换离子回旋共振-质谱法（nanoLC-FTICR-MS）进行分析，该方法在提取具有独特质量差的AOD质量对方面具有很高的特异性，并允许基于同位素比的相对定量。使用这种方法，我们量化并绘制了模型蛋白，重组人血清白蛋白（rHSA）在其天然形式（购买）和FeEDTA氧化后在蛋白质和氨基酸水平上的表面氧化位点。各种修饰的氨基酸残基，包括赖氨酸、精氨酸、脯氨酸、组氨酸、苏氨酸、天冬氨酸和谷氨酸，被发现被氧化成醛和酮的最终产物。该方法的敏感性体现在鉴定的肽的数量上，20个肽在天然蛋白上，29个在使用FeEDTA和抗坏血酸表面氧化后。所有鉴定的肽都映射到HSA晶体结构的表面，验证了该方法用于鉴定蛋白质表面氧化氨基酸。在FeEDTA氧化和天然蛋白氧化的相对定量实验中，鉴定的位点表现出不同的相对氧化倾向，不依赖于氨基酸残基。这种新颖的方法不仅具有鉴定和定量氧化蛋白的能力，而且还能对各种单个氨基酸产生位点特异性定量。我们期望将这种方法扩展到研究与疾病有关的氧化。
Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. This presents a significant advancement over current methods, combining strengths of current methods and adding the abilities to multiplex, quantitate, and probe more modified amino acids. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1,4,7,10-tetraazacyclododecane-N,N',",N'"-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to alclehyde or keto end products. O-ECAT can be loaded with a variety of metals, which yields the ability to generate mass pairs and multiplex multiple samples. The O-ECAT moiety also serves as a handle. for identification, quantitation, and affinity purification. After proteolysis, the AOD-tagged peptides are affinity purified and analyzed by nanoLC-FTICR-MS (nanoliquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry), which provides high specificity in extracting coeluting AOD mass pairs with a unique mass difference and allows relative quantitation based on isotopic ratios. Using this methodology, we have quantified and mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation both at the protein and amino acid levels. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic, and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure, validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities toward oxidation, independent of amino acid residue. This novel methodology not only has the ability to identify and quantitate oxidized proteins but also yields site-specific quantitation on a variety of individual amino acids. We expect to extend this methodology to study disease-related oxidation.