Label-free proteomic identification of endogenous, insulin-stimulated interaction partners of insulin receptor substrate-1.
Label-free proteomic identification of endogenous, insulin-stimulated interaction partners of insulin receptor substrate-1.
复制标题
DOI：
10.1007/s13361-010-0051-2
复制
发表时间：
2011-03
影响因子：
3.2
通讯作者：
Yi, Zhengping
中科院分区：
文献类型：
--
作者：
Geetha, Thangiah;Langlais, Paul;Luo, Moulun;Mapes, Rebekka;Lefort, Natalie;Chen, Shu-Chuan;Mandarino, Lawrence J.;Yi, Zhengping
关键词：
蛋白质之间的相互作用是大多数细胞过程的关键。基于串联质谱学(MS/MS)的蛋白质组学与免疫共沉淀(CO-IP)相结合已成为研究蛋白质复合体的有力手段。然而，大多数关于蛋白质-蛋白质相互作用的系统蛋白质组学研究涉及使用蛋白质过度表达和/或表位标记的诱饵蛋白，这可能会影响结合化学计量学并导致较高的假阳性。在这里，我们报告了一种直接的、无标记的CO-IP-MS/MS方法，不使用蛋白质过表达或蛋白质标签，来研究基础条件和胰岛素刺激条件下与诱饵蛋白相关的内源蛋白丰度的变化，在这种情况下，诱饵蛋白是胰岛素受体底物-1(IRS-1)。IRS-1在胰岛素信号转导通路中发挥核心作用。涉及IRS-1的蛋白质-蛋白质相互作用的缺陷可能导致胰岛素抵抗和2型糖尿病的发生。HPLC-ESI-MS/MS分析在L6肌管中发现了11个新的内源性胰岛素刺激的IRS-1相互作用伙伴，包括在蛋白质去磷酸化[蛋白磷酸酶1调节亚基12A，(PPP1R12A)]、肌肉收缩和肌动蛋白细胞骨架重排、内质网应激、蛋白质折叠和蛋白质合成中起重要作用的蛋白质。这种无标记的CO-IP-MS/MS定量方法用于评估特定蛋白质的内源性相互作用伙伴，将有助于理解各种细胞刺激如何调节胰岛素信号转导。
Protein–protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein–protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein–protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.
登录
查看更多内容
DOI：
10.1073/pnas.0912599106
发表时间：
2010-02-09
影响因子：
11.1
作者：
Malovannaya, Anna;Li, Yehua;Qin, Jun
通讯作者：
Qin, Jun
影响因子：
4.4
作者：
Foster, LJ;Rudich, A;Klip, A
通讯作者：
Klip, A
影响因子：
3.9
作者：
Proud, CG
通讯作者：
Proud, CG
影响因子：
4.8
作者：
Mothe, I;VanObberghen, E
通讯作者：
VanObberghen, E
影响因子：
46.9
作者：
Rinner, Oliver;Mueller, Lukas N.;Aebersold, Ruedi
通讯作者：
Aebersold, Ruedi