Improved identification and relative quantification of sites of peptide and protein oxidation for hydroxyl radical footprinting.
Improved identification and relative quantification of sites of peptide and protein oxidation for hydroxyl radical footprinting.
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DOI：
10.1007/s13361-013-0719-5
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发表时间：
2013-11
影响因子：
3.2
通讯作者：
Sharp, Joshua S.
中科院分区：
文献类型：
--
作者：
Li, Xiaoyan;Li, Zixuan;Xie, Boer;Sharp, Joshua S.
蛋白质氧化通常与氧化应激和衰老相关，并影响正常和病理过程中的蛋白质功能。此外，故意氧化标记用于探测羟基自由基蛋白质足迹（HRPF）中的蛋白质结构和蛋白质-配体相互作用。氧化通常发生在多个位点，导致氧化异构体的混合物，其仅在修饰位点上不同。我们利用合成的异构体“氧化”肽组来测试和比较电子转移解离（ETD）和碰撞诱导解离（CID）以及纳米超高效液相色谱（nanoUPLC）分离的能力，以定量在肽混合物中多个相邻位点处具有一个氧化的氧化异构体。通过ETD的串联质谱法生成碎片离子比率，其准确地报告特定位点上的相对氧化修饰程度，而不管前体离子的电荷状态如何。相反，发现CID仅在较高前体电荷状态下产生定量MS/MS产物离子。还鉴定了在蛋白Robo-1 Ig 1 -2（一种参与神经系统轴突引导的蛋白质）的HRPF产物中的两个肽序列中的每一个中具有多个氧化位点的氧化异构体，并且通过ETD定量每个残基处的氧化程度，而无需事先进行液相色谱（LC）分离。ETD已被证明是一种可靠的技术，用于同时识别和相对定量的各种功能不同的氧化异构体，是一个有价值的工具，用于氧化应激的研究，以及提高空间分辨率的HRPF研究。
Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein-ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric “oxidized” peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.
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影响因子：
2.3
作者：
Srikanth, Rapole;Wilson, Jonathan;Vachet, Richard W.
通讯作者：
Vachet, Richard W.
DOI：
10.1016/j.jasms.2008.07.013
发表时间：
2008-11-01
影响因子：
3.2
作者：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
3.7
作者：
Gitlin, G;Tsarbopoulos, A;Bausch, JN
通讯作者：
Bausch, JN