Analysis of Human Nuclear Protein Complexes by Quantitative Mass Spectrometry Profiling.
Analysis of Human Nuclear Protein Complexes by Quantitative Mass Spectrometry Profiling.
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DOI：
10.1002/pmic.201700427
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发表时间：
2018-06
期刊：
影响因子：
3.4
通讯作者：
Aryal UK
中科院分区：
文献类型：
--
作者：
Connelly KE;Hedrick V;Paschoal Sobreira TJ;Dykhuizen EC;Aryal UK
蛋白质复合物的分析提供了对表达的蛋白质组的集合如何被组织成功能单元的见解。虽然细胞质蛋白质复合物的蛋白质组范围分析技术已经取得了进展，但有关人类核蛋白质复合物的信息非常有限。为了缩小这一差距，我们结合天然尺寸排阻色谱法（SEC）与无标记的定量MS分析，以表征数百个从人多形性胶质母细胞瘤T98 G细胞分离的核蛋白复合物。我们确定了1794个蛋白质，其中1244个蛋白质的特点是存在于稳定相关的推定复合物之间的两个生物重复重叠。co-IP实验证实了PARP 1与Ku 70/Ku 80蛋白和HDAC 1（组蛋白脱乙酰酶复合物1）和CHD 4的相互作用。HDAC 1/2还与SEC分级分离中的各种SIN 3A和核小体重塑和脱乙酰酶组分共迁移，包括SIN 3A、SAP 30、RBBP 4、RBBP 7和NCOR 1。HDAC 1/2/3与KDM 1A和RCOR 1的共洗脱进一步证实了这些蛋白质是人脱乙酰酶复合物的组成部分。我们的方法还证明了识别潜在的兼职复合物和含有未表征蛋白质的新型复合物的能力。总体而言，结果证明了SEC分级分离和LC-MS分析用于蛋白质的系统范围分析以预测不同形式的核蛋白复合物的存在的效用。
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.
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