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改进的肽和蛋白质氧化位点的鉴定和相对定量用于羟自由基足迹法。

Improved identification and relative quantification of sites of peptide and protein oxidation for hydroxyl radical footprinting.

机构信息

Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602, USA.

出版信息

J Am Soc Mass Spectrom. 2013 Nov;24(11):1767-76. doi: 10.1007/s13361-013-0719-5. Epub 2013 Sep 7.

Abstract

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.

摘要

蛋白质氧化通常与氧化应激和衰老有关,并影响正常和病理过程中的蛋白质功能。此外,故意的氧化标记被用于探测蛋白质结构和蛋白质-配体相互作用在羟基自由基蛋白质足迹(HRPF)。氧化通常发生在多个部位,导致修饰部位不同的氧化异构体混合物。我们利用一组合成的、异构的“氧化”肽来测试和比较电子转移解离(ETD)和碰撞诱导解离(CID)的能力,以及纳米超高效液相色谱(nanoUPLC)分离,以定量混合物中多个相邻部位的氧化异构体肽。通过 ETD 的串联质谱生成的片段离子比准确报告特定部位的相对氧化修饰程度,而与前体离子的电荷状态无关。相反,CID 被发现仅在较高的前体电荷状态下生成定量的 MS/MS 产物离子。在蛋白质 Robo-1 Ig1-2 的 HRPF 产物中的两个肽序列中的每个序列中具有多个氧化部位的氧化异构体也被鉴定出来,并且每个残基的氧化程度通过 ETD 进行定量,而无需预先进行液相色谱(LC)分离。ETD 已被证明是一种可靠的技术,可用于同时鉴定和相对定量各种功能不同的氧化异构体,并且是研究氧化应激以及提高 HRPF 研究的空间分辨率的有用工具。

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