Solid-phase capture for the detection and relative quantification of S-nitrosoproteins by mass spectrometry.
Solid-phase capture for the detection and relative quantification of S-nitrosoproteins by mass spectrometry.
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DOI：
10.1016/j.ymeth.2012.10.001
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发表时间：
2013-08-01
期刊：
影响因子：
4.8
通讯作者：
Foster, Matthew W.
中科院分区：
文献类型：
--
作者：
Thompson, J. Will;Forrester, Michael T.;Moseley, M. Arthur;Foster, Matthew W.
关键词：
S-亚硝基化蛋白（SNO-蛋白）的蛋白质组学分析长期以来依赖于生物素开关技术（BST），该技术需要封闭游离巯基，基于抗坏血酸的SNO-Cys的脱亚硝基化，新生巯基的生物素化和基于亲和素的亲和分离。最近的发展是SNO蛋白的树脂辅助捕获（SNO-RAC），其用硫丙基琼脂糖（TPS）代替生物素-抗生物素蛋白，从而减少了富集S-亚硝基化蛋白所需的步骤数量。此外，SNO-RAC促进在SNO-蛋白捕获后的树脂上蛋白水解消化，大大简化了含有S-亚硝基化位点（“SNO-位点”）的肽的纯化。这种基于树脂的方法现在也被应用于检测替代的基于Cys的修饰，包括S-棕榈酰化（Acyl-RAC）和S-氧化（Ox-RAC）。在这里，我们回顾了重要的步骤，以尽量减少假阳性识别的SNO蛋白，给出了详细的方法处理蛋白结合TPS的质谱（MS）为基础的分析，并讨论了各种定量MS方法，是兼容的SNO-RAC。我们还讨论了策略，以克服目前的局限性周围MS为基础的SNO-网站本地化的肽含有一个以上的潜在目标半胱氨酸残基。因此，这篇文章作为一个出发点和指导MS为重点的探索SNO-蛋白质组的SNO-RAC。
The proteomic analysis of S-nitrosylated protein (SNO-proteins) has long depended on the biotin switch technique (BST), which requires blocking of free thiols, ascorbate-based denitrosylation of SNO-Cys, biotinylation of nascent thiol and avidin-based affinity isolation. A more recent development is resin assisted-capture of SNO-proteins (SNO-RAC), which substitutes thiopropyl sepharose (TPS) for biotin-avidin, thus reducing the number of steps required for enrichment of S-nitrosylated proteins. In addition, SNO-RAC facilitates on-resin proteolytic digestion following SNO-protein capture, greatly simplifying the purification of peptides containing sites of S-nitrosylation (“SNO-sites”). This resin-based approach has also now been applied to detection of alternative Cys-based modifications, including S-palmitoylation (Acyl-RAC) and S-oxidation (Ox-RAC). Here, we review the important steps to minimize false-positive identification of SNO-proteins, give detailed methods for processing of protein-bound TPS for mass spectrometry (MS) based analysis, and discuss the various quantitative MS methods that are compatible with SNO-RAC. We also discuss strategies to overcome the current limitations surrounding MS-based SNO-site localization in peptides containing more than one potential target Cys residue. This article therefore serves as a starting point and guide for the MS-focused exploration of SNO-proteomes by SNO-RAC.
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影响因子：
168.9
作者：
Moya, MP;Gow, AJ;Stamler, JS
通讯作者：
Stamler, JS
DOI：
10.1073/pnas.1008036107
发表时间：
2010-09-28
影响因子：
11.1
作者：
Doulias, Paschalis-Thomas;Greene, Jennifer L.;Ischiropoulos, Harry
通讯作者：
Ischiropoulos, Harry
影响因子：
4.8
作者：
Forrester, Michael T.;Foster, Matthew W.;Stamler, Jonathan S.
通讯作者：
Stamler, Jonathan S.
影响因子：
2.9
作者：
Benhar, Moran;Thompson, J. Will;Moseley, M. Arthur;Stamler, Jonathan S.
通讯作者：
Stamler, Jonathan S.
影响因子：
13.6
作者：
Foster MW;Hess DT;Stamler JS
通讯作者：
Stamler JS