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An Optimized Chromatographic Strategy for Multiplexing In Parallel Reaction Monitoring Mass Spectrometry: Insights from Quantitation of Activated Kinases

机译:并行反应监测质谱中多路复用的最佳色谱策略:活化激酶定量分析的启示

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摘要

Reliable quantitation of protein abundances in defined sets of cellular proteins is critical to numerous biological applications. Traditional immunodetection-based methods are limited by the quality and availability of specific antibodies, especially for site-specific post-translational modifications. Targeted proteomic methods, including the recently developed parallel reaction monitoring (PRM) mass spectrometry, have enabled accurate quantitative measurements of up to a few hundred specific target peptides. However, the degree of practical multiplexing in label-free PRM workflows remains a significant limitation for the technique. Here we present a strategy for significantly increasing multiplexing in label-free PRM that takes advantage of the superior separation characteristics and retention time stability of meter-scale monolithic silica-C18 column-based chromatography. We show the utility of the approach in quantifying kinase abundances downstream of previously developed active kinase enrichment methodology based on multidrug inhibitor beads. We examine kinase activation dynamics in response to three different MAP kinase inhibitors in colorectal carcinoma cells and demonstrate reliable quantitation of over 800 target peptides from over 150 kinases in a single label-free PRM run. The kinase activity profiles obtained from these analyses reveal compensatory activation of TGF-β family receptors as a response to MAPK blockade. The gains achieved using this label-free PRM multiplexing strategy will benefit a wide array of biological applications.
机译:对确定的细胞蛋白质组中的蛋白质丰度进行可靠的定量分析对于许多生物学应用而言至关重要。传统的基于免疫检测的方法受到特异性抗体的质量和可用性的限制,特别是针对位点特异性翻译后修饰。靶向蛋白质组学方法,包括最近开发的平行反应监测(PRM)质谱,已经能够精确定量测量多达数百种特定的目标肽。但是,无标签PRM工作流程中的实用复用程度仍然是对该技术的重大限制。在这里,我们提出了一种显着提高无标记PRM中多路复用的策略,该策略利用了米级整体硅胶C18色谱柱色谱的优异分离特性和保留时间稳定性。我们显示了该方法在量化基于多药抑制剂珠的先前开发的活性激酶富集方法下游的激酶丰度方面的实用性。我们检查了三种不同的MAP激酶抑制剂在大肠癌细胞中的激酶激活动力学,并证明了在无标签的PRM运行中从150多种激酶中可靠地定量了800多种目标肽。从这些分析中获得的激酶活性图谱显示,TGF-β家族受体的代偿性激活是对MAPK阻断的反应。使用这种无标记PRM复用策略所获得的收益将使广泛的生物学应用受益。

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