Selective chemoprecipitation to enrich nitropeptides from complex proteomes for mass-spectrometric analysis.
Selective chemoprecipitation to enrich nitropeptides from complex proteomes for mass-spectrometric analysis.
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DOI：
10.1038/nprot.2014.052
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发表时间：
2014-04
期刊：
影响因子：
14.8
通讯作者：
Prokai-Tatrai K
中科院分区：
文献类型：
--
作者：
Prokai L;Guo J;Prokai-Tatrai K
翻译后蛋白质硝化由于其参与细胞信号传导、影响蛋白质功能以及作为氮氧化应激的潜在生物标志物而引起人们的兴趣。我们描述了一种用于富集硝基肽的基于质谱的蛋白质组学的方法，该方法是基于免疫亲和性的方法的简单而可靠的替代方法。该方法的起始材料是蛋白水解消化物。将肽与甲醛和氰基硼氢化钠反应以使所有N-末端和侧链氨基二甲基化。随后加入连二亚硫酸钠以将硝基还原为胺;理论上，存在的唯一氨基最初将是硝基。然后将肽样品应用于固相活性酯试剂，并且那些具有氨基的肽将被选择性地和共价地捕获。在用三氟乙酸水解时肽的释放产生具有4-甲酰基-苯甲酰氨基的肽，其中硝基曾经是。在定性设置中，该程序可用于识别由活性氮物种修饰的蛋白质，并确定其硝化的特定位点。定量测量可以通过在还原二甲基化步骤中对肽进行稳定同位素标记来进行。固相活性酯试剂的制备需要约1天。硝基肽的富集需要约2天，样品制备需要1至30小时，根据实验设计而有所不同。LC-MS/MS分析需要4小时至数天，数据处理可以在1至7天内完成。
Posttranslational protein nitration has attracted interest due to its involvement in cellular signaling, effects on protein function, and as a potential biomarker of nitroxidative stress. We describe a procedure for enriching nitropeptides for mass spectrometry-based proteomics that is a simple and reliable alternative to immunoaffinity-based methods. The starting material for this procedure is a proteolytic digest. The peptides are reacted with formaldehyde and sodium cyanoborohydride to dimethylate all the N-terminal and side-chain amino groups. Sodium dithionite is added subsequently to reduce the nitro groups to amines; in theory, the only amino groups present will have originally been nitro groups. The peptide sample is then applied to a solid-phase active ester reagent, and those peptides with amino groups will be selectively and covalently captured. Release of the peptides on hydrolysis with trifluoroacetic acid results in peptides that have a 4-formyl-benzamido group where the nitro group used to be. In qualitative setups, the procedure can be used to identify proteins modified by reactive nitrogen species and to determine the specific sites of their nitration. Quantitative measurements can be performed by stable-isotope labeling of the peptides in the reductive dimethylation step. Preparation of the solid-phase active ester reagent takes about 1 day. Enrichment of nitropeptides requires about 2 days, and sample preparations need 1 to 30 h varying based on experimental design. LC–MS/MS assays take from 4 h to several days and data processing can be done 1 to 7 days.