Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 201 S. University Street, 47907, West Lafayette, IN, USA.
Purdue Proteomics Facility, Bindley Biosciences Center, Discovery Park, Purdue University, 1203 W. State Street, 47907, West Lafayette, IN, USA.
Proteomics. 2018 Jun;18(11):e1700427. doi: 10.1002/pmic.201700427. Epub 2018 May 4.
Analysis of protein complexes provides insights into how the ensemble of expressed proteome is organized into functional units. While there have been advances in techniques for proteome-wide profiling of cytoplasmic protein complexes, information about human nuclear protein complexes are very limited. To close this gap, we combined native size exclusion chromatography (SEC) with label-free quantitative MS profiling to characterize hundreds of nuclear protein complexes isolated from human glioblastoma multiforme T98G cells. We identified 1794 proteins that overlapped between two biological replicates of which 1244 proteins were characterized as existing within stably associated putative complexes. co-IP experiments confirmed the interaction of PARP1 with Ku70/Ku80 proteins and HDAC1 (histone deacetylase complex 1) and CHD4. HDAC1/2 also co-migrated with various SIN3A and nucleosome remodeling and deacetylase components in SEC fractionation including SIN3A, SAP30, RBBP4, RBBP7, and NCOR1. Co-elution of HDAC1/2/3 with both the KDM1A and RCOR1 further confirmed that these proteins are integral components of human deacetylase complexes. Our approach also demonstrated the ability to identify potential moonlighting complexes and novel complexes containing uncharacterized proteins. Overall, the results demonstrated the utility of SEC fractionation and LC-MS analysis for system-wide profiling of proteins to predict the existence of distinct forms of nuclear protein complexes.
蛋白质复合物的分析提供了深入了解表达蛋白质组如何组织成功能单元的线索。虽然在细胞质蛋白质复合物的全蛋白质组分析技术方面已经取得了进展，但关于人类核蛋白质复合物的信息却非常有限。为了弥补这一差距，我们结合了天然大小排阻色谱（SEC）和无标记定量 MS 分析，以表征从人类多形性胶质母细胞瘤 T98G 细胞中分离的数百种核蛋白质复合物。我们鉴定了两个生物学重复之间重叠的 1794 种蛋白质，其中 1244 种蛋白质被鉴定为存在于稳定相关的假定复合物中。co-IP 实验证实了 PARP1 与 Ku70/Ku80 蛋白和 HDAC1（组蛋白去乙酰化酶复合物 1）和 CHD4 的相互作用。HDAC1/2 也与 SEC 分级分离中的各种 SIN3A 和核小体重塑和去乙酰化酶成分共迁移，包括 SIN3A、SAP30、RBBP4、RBBP7 和 NCOR1。HDAC1/2/3 与 KDM1A 和 RCOR1 的共洗脱进一步证实了这些蛋白质是人类去乙酰化酶复合物的组成部分。我们的方法还证明了识别潜在的月光复合物和包含未鉴定蛋白质的新型复合物的能力。总的来说，这些结果证明了 SEC 分级分离和 LC-MS 分析用于全系统蛋白质分析以预测不同形式的核蛋白质复合物的存在的实用性。
