On-beads digestion in conjunction with data-dependent mass spectrometry: a shortcut to quantitative and dynamic interaction proteomics.
On-beads digestion in conjunction with data-dependent mass spectrometry: a shortcut to quantitative and dynamic interaction proteomics.
复制标题
与数据依赖性质谱法结合使用的珠子消化：定量和动态相互作用蛋白质组学的快捷方式。
DOI：
10.3390/biology3020320
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发表时间：
2014-04-16
期刊：
影响因子：
4.2
通讯作者：
von Kriegsheim A
中科院分区：
文献类型：
--
作者：
Turriziani B;Garcia-Munoz A;Pilkington R;Raso C;Kolch W;von Kriegsheim A
随着“组学”时代的到来，生物学研究已经从分析单个蛋白质的功能转向了解整个蛋白质网络如何连接和适应环境线索。通常，病理过程是由一个功能失调的蛋白质网络而不是单个蛋白质引发的。因此，研究蛋白质在途径和信号网络背景下的调控是至关重要的。在这项研究中，我们证明了定量相互作用蛋白质组学方法，结合免疫沉淀，溶液内消化和无标记定量质谱，提供了高精度和深度的数据。该方案适用于标记的外源性和未标记的内源性蛋白质。此外，它是快速，可靠的，并且由于无标记的定量方法，允许多种条件的比较。我们进一步表明，我们能够以中等吞吐量的方式生成数据，并且我们可以量化信号通路中响应有丝分裂刺激的动态相互作用变化，使我们的方法成为生成系统生物学方法数据的合适方法。
With the advent of the “-omics” era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a single protein. It is therefore crucial to investigate the regulation of proteins in the context of a pathway first and signaling network second. In this study, we demonstrate that a quantitative interaction proteomic approach, combining immunoprecipitation, in-solution digestion and label-free quantification mass spectrometry, provides data of high accuracy and depth. This protocol is applicable, both to tagged, exogenous and untagged, endogenous proteins. Furthermore, it is fast, reliable and, due to a label-free quantitation approach, allows the comparison of multiple conditions. We further show that we are able to generate data in a medium throughput fashion and that we can quantify dynamic interaction changes in signaling pathways in response to mitogenic stimuli, making our approach a suitable method to generate data for system biology approaches.