Confident identification of 3-nitrotyrosine modifications in mass spectral data across multiple mass spectrometry platforms.
Confident identification of 3-nitrotyrosine modifications in mass spectral data across multiple mass spectrometry platforms.
复制标题
跨多个质谱平台可靠地鉴定质谱数据中的 3-硝基酪氨酸修饰。
DOI：
10.1016/j.jprot.2011.04.007
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发表时间：
2011
影响因子：
3.3
通讯作者：
Gibson,BradfordW
中科院分区：
文献类型：
--
作者：
Li,Bensheng;Held,JasonM;Schilling,Birgit;Danielson,StevenR;Gibson,BradfordW
3-硝基酪氨酸 (3NT) 是一种与许多疾病相关的氧化翻译后修饰。由于生物样品中 3NT 修饰的化学计量较低，确定这种修饰的具体位点仍然是一个挑战。基于质谱的蛋白质组学是识别 3NT 修饰的强大工具，但后来证明识别 3NT 位点的几份报告是不正确的，这突出表明这些工作流程的准确性和效率都需要改进。为了加深我们对含 3NT 肽的色谱和光谱特性的理解，我们采用了一种简单、可重复的程序，通过对定义的市售 48 种蛋白质混合物进行化学硝化来生成大量 3NT 肽。使用两个互补的 LC-MS/MS 平台、QTOF (QSTAR Elite) 和双压离子阱质谱仪 (LTQ Velos)，我们检测到了 200 多种经过验证的含 3NT 肽，两个系统检测到的肽有显着重叠。我们使用定义的标准手动研究了每种肽的 LC-MS/MS 特性，然后评估其效用以确认该肽经过 3NT 修饰。这套经过验证的含 3NT 肽可用于优化质谱仪器和数据挖掘策略，或进一步开发针对这种生物学上重要的氧化翻译后修饰的 3NT 肽富集策略。
3-nitrotyrosine (3NT) is an oxidative posttranslational modification associated with many diseases. Determining the specific sites of this modification remains a challenge due to the low stoichiometry of 3NT modifications in biological samples. Mass spectrometry-based proteomics is a powerful tool for identifying 3NT modifications, however several reports identifying 3NT sites were later demonstrated to be incorrect, highlighting that both the accuracy and efficiency of these workflows need improvement. To advance our understanding of the chromatographic and spectral properties of 3NT-containing peptides we have adapted a straightforward, reproducible procedure to generate a large set of 3NT peptides by chemical nitration of a defined, commercially available 48 protein mixture. Using two complementary LC–MS/MS platforms, a QTOF (QSTAR Elite) and dual pressure ion trap mass spectrometer (LTQ Velos), we detected over 200 validated 3NT-containing peptides with significant overlap in the peptides detected by both systems. We investigated the LC–MS/MS properties for each peptide manually using defined criteria and then assessed their utility to confirm that the peptide was 3NT modified. This broad set of validated 3NT-containing peptides can be utilized to optimize mass spectrometric instrumentation and data mining strategies or further develop 3NT peptide enrichment strategies for this biologically important, oxidative posttranslational modification.