Accurate Identification of Deamidation and Citrullination from Global Shotgun Proteomics Data Using a Dual-Search Delta Score Strategy.
Accurate Identification of Deamidation and Citrullination from Global Shotgun Proteomics Data Using a Dual-Search Delta Score Strategy.
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使用双搜索 Delta 评分策略从全球鸟枪法蛋白质组数据中准确识别脱酰胺和瓜氨酸。
DOI：
10.1021/acs.jproteome.9b00766
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发表时间：
2020
影响因子：
4.4
通讯作者：
Qian,Wei-Jun
中科院分区：
文献类型：
--
作者：
Wang,Xi;Swensen,AdamC;Zhang,Tong;Piehowski,PaulD;Gaffrey,MatthewJ;Monroe,MatthewE;Zhu,Ying;Dong,Hailiang;Qian,Wei-Jun
具有脱酰胺/瓜氨酸氨基酸的蛋白质在许多人类疾病的发病机制中发挥着关键作用；然而，识别复杂生物样品中的这些修饰一直是一个持续的挑战。在此，我们提出了一种方法，可以从通过激光捕获显微切割获得的人类胰岛的深度蛋白质组分析研究生成的鸟枪蛋白质组学数据中准确识别这些修饰。使用 MSGF+ 数据库匹配对所有 MS/MS 谱图进行两次搜索，对氨基酸天冬酰胺、谷氨酰胺和精氨酸 (NQR) 进行或不进行动态 +0.9840 Da 质量位移修饰。因此，每个谱图都会生成两个具有 MSGF+ 分数的肽谱匹配 (PSM)，用于 Delta 分数计算。据观察，所有具有正 Delta 得分值的 PSM 都聚集在一起，质量误差在 0 ppm 左右，而具有负 Delta 得分值的 PSM 在数据库搜索的定义质量误差范围 (20 ppm) 内几乎均匀分布。为了估计修饰肽的错误发现率 (FDR)，应用了“目标模拟”策略，其中根据包含“真实修饰”(+0.9840 Da) 和“模拟修饰”(+1.0227 Da) 肽质量的串联数据库搜索数据集。使用大于零的 Delta Score 过滤器值将 FDR 控制在~2%。手动检查光谱表明，具有正 Delta Score 值的 PSM 在其 MS/MS 光谱中包含脱酰胺/瓜氨酸片段。本研究中鉴定的许多瓜氨酸位点在文献中被生物化学证实为自身免疫性疾病的自身免疫原性表位。结果表明，使用这种双搜索 Delta Score 策略，可以从鸟枪法组织蛋白质组数据中准确识别原位脱酰胺/瓜氨酸肽。原始 MS 数据可在 ProteomeXchange (PXD010150) 上获取。
Proteins with deamidated/citrullinated amino acids play critical roles in the pathogenesis of many human diseases; however, identifying these modifications in complex biological samples has been an ongoing challenge. Herein we present a method to accurately identify these modifications from shotgun proteomics data generated by a deep proteome profiling study of human pancreatic islets obtained by laser capture microdissection. All MS/MS spectra were searched twice using MSGF+ database matching, with and without a dynamic +0.9840 Da mass shift modification on amino acids asparagine, glutamine, and arginine (NQR). Consequently, each spectrum generates two peptide-to-spectrum matches (PSMs) with MSGF+ scores, which were used for the Delta Score calculation. It was observed that all PSMs with positive Delta Score values were clustered with mass errors around 0 ppm, while PSMs with negative Delta Score values were distributed nearly equally within the defined mass error range (20 ppm) for database searching. To estimate false discovery rate (FDR) of modified peptides, a “target-mock” strategy was applied in which data sets were searched against a concatenated database containing “real-modified” (+0.9840 Da) and “mock-modified” (+1.0227 Da) peptide masses. The FDR was controlled to ∼2% using a Delta Score filter value greater than zero. Manual inspection of spectra showed that PSMs with positive Delta Score values contained deamidated/citrullinated fragments in their MS/MS spectra. Many citrullinated sites identified in this study were biochemically confirmed as autoimmunogenic epitopes of autoimmune diseases in literature. The results demonstrated that in situ deamidated/citrullinated peptides can be accurately identified from shotgun tissue proteomics data using this dual-search Delta Score strategy. Raw MS data is available at ProteomeXchange (PXD010150).