Protein cross-linking capillary electrophoresis at increased throughput for a range of protein-protein interactions.
Protein cross-linking capillary electrophoresis at increased throughput for a range of protein-protein interactions.
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DOI：
10.1039/c7an02098h
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发表时间：
2018-04-16
期刊：
影响因子：
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通讯作者：
Kennedy RT
中科院分区：
文献类型：
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作者：
Ouimet CM;Dawod M;Grinias J;Assimon VA;Lodge J;Mapp AK;Gestwicki JE;Kennedy RT
用于测量蛋白质-蛋白质复合物的亲和力和化学计量的工具对于阐明蛋白质-蛋白质相互作用（PPI）在控制细胞功能和筛选PPI调节剂中的作用是有价值的。这种测量可能具有挑战性，因为PPI可以跨越广泛的亲和力，并包括从二聚体到高阶低聚物的化学计量。此外，大多数技术需要大量的蛋白质，这可能会阻碍难以获得蛋白质的研究。蛋白质交联毛细管电泳（PXCE）有可能直接测量PPI，甚至在消耗阿托摩尔量的同时分辨多个PPI。以前PXCE仅用于高亲和力，1：1复合物;在这里，我们扩展PXCE的效用，以获得广泛的PPI，包括弱和多聚体寡聚体。使用戊二醛作为交联剂是推进该方法的关键，因为它具有快速的反应动力学。发现10 s的反应时间足以交联和定量7种不同PPI，其Kd值范围从低μM到低nM，包括与热休克组织蛋白（3.8 ± 0.7 μM）和bcl 2相关anthanogene（26 ± 6 nM）相互作用的热休克蛋白70（Hsp 70）。通过尺寸排阻色谱法评估，发现蛋白质浓度< 20 μM时，蛋白质聚集体的非特异性交联最小。PXCE足够灵敏，可以测量由蛋白质核苷酸状态或蛋白质结合位点点突变诱导的PPI亲和力变化。此外，几种相互作用可以在单次运行中解决，包括Hsp 70单体，同源二聚体和Hsp 70与Hsp 70相互作用蛋白（CHIP）的C-末端复合。最后，PXCE的通量增加到每个样品1分钟，表明在筛选中的实用性的潜力。
Tools for measuring affinities and stoichiometries of protein-protein complexes are valuable for elucidating the role of protein-protein interactions (PPIs) in governing cell functions and screening for PPI modulators. Such measurements can be challenging because PPIs can span a wide range of affinities and include stoichiometries from dimers to high order oligomers. Also, most techniques require large amounts of protein which can hamper research for difficult to obtain proteins. Protein cross-linking capillary electrophoresis (PXCE) has the potential to directly measure PPIs and even resolve multiple PPIs while consuming attomole quantities. Previously PXCE has only been used for high affinity, 1:1 complexes; here we expand the utility of PXCE to access a wide range of PPIs including weak and multimeric oligomers. Use of glutaraldehyde as the cross-linking agent was key to advancing the method because of its rapid reaction kinetics. A 10 s reaction time was found to be sufficient for cross-linking and quantification of seven different PPIs with Kd values ranging from low μM to low nM including heat shock protein 70 (Hsp70) interacting with heat shock organizing protein (3.8 ± 0.7 μM) and bcl2 associated anthanogene (26 ± 6 nM). Non-specific cross-linking of protein aggregates was found to be minimal at protein concentrations < 20 μM as assessed by size exclusion chromatography. PXCE was sensitive enough to measure changes in PPI affinity induced by the protein nucleotide state or point mutations in the protein-binding site. Further, several interactions could be resolved in a single run, including Hsp70 monomer, homodimer and Hsp70 complexed the with c-terminus of Hsp70 interacting protein (CHIP). Finally, the throughput of PXCE was increased to 1 min per sample suggesting potential for utility in screening.