A solid phase extraction-based platform for rapid phosphoproteomic analysis.
A solid phase extraction-based platform for rapid phosphoproteomic analysis.
复制标题
基于固相萃取的快速磷酸蛋白质组分析平台。
DOI：
10.1016/j.ymeth.2011.03.008
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发表时间：
2011-08
期刊：
影响因子：
4.8
通讯作者：
Gygi, Steven P.
中科院分区：
文献类型：
--
作者：
Dephoure, Noah;Gygi, Steven P.
蛋白质磷酸化是最常见和深入研究的翻译后蛋白质修饰之一。它几乎在所有细胞过程中发挥着至关重要的作用，并与包括癌症在内的许多人类疾病有关。用于识别和监测磷酸化位点的传统生化和遗传学方法费力且缓慢，近年来已大部分被质谱分析所取代。改进的磷酸肽富集方法加上更快、更灵敏的质谱仪导致磷酸蛋白质组数据集的规模激增。然而，这些方法的更广泛应用受到设备成本和由此产生的对仪器时间的高需求以及生物学家和质谱学家之间的技术差距的限制。在这里，我们描述了一种改进的两步富集策略，该策略采用 lysC 消化和自装强阳离子交换 (SCX) 固相萃取 (SPE) 柱的逐步洗脱，然后使用混合 LTQ Orbitrap Velos 质谱仪进行固定金属离子亲和色谱 (IMAC) 和 LC-MS/MS 分析。 SCX 程序不需要 HPLC 系统，几乎不需要专业知识，而且由于可以并行处理多个样品，因此可以节省大量时间和劳动力。我们证明了这种方法与稳定同位素标记相结合，可以在 12 小时的仪器分析时间内量化酵母中含有超过 8,000 个独特磷酸化位点的肽，并检查酶选择和仪器平台的影响。
Protein phosphorylation is among the most common and intensely studied post-translational protein modification. It plays crucial roles in virtually all cellular processes and has been implicated in numerous human diseases, including cancer. Traditional biochemical and genetic methods for identifying and monitoring sites of phosphorylation are laborious and slow and in recent years have largely been replaced by mass spectrometric analysis. Improved methods for phosphopeptide enrichment coupled with faster and more sensitive mass spectrometers have led to an explosion in the size of phosphoproteomic datasets. However, wider application of these methods is limited by equipment costs and the resultant high demand for instrument time as well as by a technology gap between biologists and mass spectrometrists. Here we describe a modified two-step enrichment strategy that employs lysC digestion and step elution from self-packed strong cation exchange (SCX) solid phase extraction (SPE) columns followed by immobilized metal ion affinity chromatography (IMAC) and LC-MS/MS analysis using a hybrid LTQ Orbitrap Velos mass spectrometer. The SCX procedure does not require an HPLC system, demands little expertise, and because multiple samples can be processed in parallel, can provide a large savings of time and labor. We demonstrate this method in conjunction with stable isotope labeling to quantify peptides harboring >8,000 unique phosphorylation sites in yeast in 12 hours of instrument analysis time and examine the impact of enzyme choice and instrument platform.
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影响因子：
64.5
作者：
Huttlin EL;Jedrychowski MP;Elias JE;Goswami T;Rad R;Beausoleil SA;Villén J;Haas W;Sowa ME;Gygi SP
通讯作者：
Gygi SP
影响因子：
4.4
作者：
Bakalarski, Corey E.;Elias, Joshua E.;Villen, Judit;Haas, Wilhelm;Gerber, Scott A.;Everley, Patrick A.;Gygi, Steven P.
通讯作者：
Gygi, Steven P.
影响因子：
7
作者：
Trinidad, JC;Specht, CG;Burlingame, AL
通讯作者：
Burlingame, AL
影响因子：
46.9
作者：
Rush, J;Moritz, A;Comb, MJ
通讯作者：
Comb, MJ
影响因子：
46.9
作者：
Ficarro, SB;McCleland, ML;White, FM
通讯作者：
White, FM