Nanodiscs and SILAC-Based Mass Spectrometry to Identify a Membrane Protein Interactome
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- 作者：Xiao X. Zhang ; Catherine S. Chan ; Huan Bao ; Yuan Fang ; Leonard J. Foster ; Franck Duong
- 刊名：Journal of Proteome Research
- 出版年：2012
- 出版时间：February 3, 2012
- 年：2012
- 卷：11
- 期：2
- 页码：1454-1459
- 全文大小：268K
- 年卷期：v.11,no.2(February 3, 2012)
- ISSN：1535-3907
文摘
Integral membrane proteins are challenging to work with biochemically given their insoluble nature; the nanodisc circumvents the difficulty by stabilizing them in small patches of lipid bilayer. Here, we show that nanodiscs combined with SILAC-based quantitative proteomics can be used to identify the soluble interacting partners of virtually any membrane protein. As a proof of principle, we applied the method to the bacterial SecYEG protein-conducting channel, the maltose transporter MalFGK2 and the membrane integrase YidC. In contrast to the detergent micelles, which tend to destabilize interactions, the nanodisc was able to capture out of a complex whole cell extract the proteins SecA, Syd, and MalE with a high degree of confidence and specificity. The method was sensitive enough to isolate these interactors as a function of the lipid composition in the disc and the culture conditions. In agreement with a previous photo-cross linking analysis, YidC did not show any high-affinity interactions with cytosolic or periplasmic proteins. These three examples illustrate the utility of nanoscale lipid bilayers to identify the soluble peripheral partners of proteins intergrated in the lipid bilayer.