Supercharging by m-NBA Improves ETD-Based Quantification of Hydroxyl Radical Protein Footprinting.
Supercharging by m-NBA Improves ETD-Based Quantification of Hydroxyl Radical Protein Footprinting.
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DOI：
10.1007/s13361-015-1129-7
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发表时间：
2015-08
影响因子：
3.2
通讯作者：
Sharp JS
中科院分区：
文献类型：
--
作者：
Li X;Li Z;Xie B;Sharp JS
羟基自由基蛋白质足迹 (HRPF) 是一种基于 MS 的技术，通过测量溶液中扩散的羟基自由基对氨基酸侧链的氧化来分析蛋白质结构。 HRPF 的空间分辨率受到可以准确定量氧化量的蛋白质最小部分的限制。先前的工作表明电子转移解离 (ETD) 是量化肽氧化异构体混合物中每个氨基酸侧链氧化量的最可靠方法，但有效的 ETD 需要高肽电荷态，这限制了其在 HRPF 中的适用性。增压试剂已被用于增强 ETD 分析中的肽电荷态，但之前的工作表明增压试剂对不同肽序列的电荷态增强效果不同；目前尚不清楚不同的氧化异构体是否会经历不同的电荷增强效果。在这里，我们报告了间硝基苯甲醇 (m-NBA) 对基于 ETD 的肽氧化定量的影响。将 m-NBA 添加到经过 HRPF 处理的合成异构氧化肽和 Robo1 蛋白的确定混合物中，增加了较高电荷态离子的丰度，从而提高了我们对混合物进行有效 ETD 的能力。在存在或不存在 m-NBA 的情况下，通过 ETD 报告的定量没有发现差异，这表明所有氧化异构体的电荷增强程度相似。这些结果表明 m-NBA 可用于 HRPF 和其他应用中肽氧化的残留水平定量。
Hydroxyl radical protein footprinting (HRPF) is an MS-based technique for analyzing protein structure based on measuring the oxidation of amino acid side chains by hydroxyl radicals diffusing in solution. Spatial resolution of HRPF is limited by the smallest portion of the protein for which oxidation amounts can be accurately quantitated. Previous work has shown electron transfer dissociation (ETD) to be the most reliable method for quantifying the amount of oxidation of each amino acid side chain in a mixture of peptide oxidation isomers, but efficient ETD requires high peptide charge states which limits its applicability for HRPF. Supercharging reagents have been used to enhance peptide charge state for ETD analysis, but previous work have shown supercharging reagents to enhance charge state differently for different peptides sequences; it is currently unknown if different oxidation isomers will experience different charge enhancement effects. Here, we report the effect of m-nitrobenzyl alcohol (m-NBA) on the ETD-based quantification of peptide oxidation. The addition of m-NBA to both a defined mixture of synthetic isomeric oxidized peptides and Robo1 protein subjected to HRPF increased the abundance of higher charge state ions, improving our ability to perform efficient ETD of the mixture. No differences in the reported quantitation by ETD were noted in the presence or absence of m-NBA, indicating that all oxidation isomers were charge-enhanced to a similar extent. These results indicate the utility of m-NBA for residue-level quantification of peptide oxidation in HRPF and other applications.