Systematic Identification of Protein Phosphorylation-Mediated Interactions.
Systematic Identification of Protein Phosphorylation-Mediated Interactions.
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蛋白质磷酸化介导的相互作用的系统性鉴定
DOI：
10.1021/acs.jproteome.0c00750
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发表时间：
2021-02-05
影响因子：
4.4
通讯作者：
Marcotte EM
中科院分区：
文献类型：
--
作者：
Floyd BM;Drew K;Marcotte EM
蛋白质磷酸化是几乎所有真核细胞过程中的关键调控机制。越来越敏感的质谱方法已经确定了数十万个磷酸化位点，但这些位点中的绝大多数的功能仍然未知，目前只有不到5%的位点被指定了功能。为了增加我们对功能性蛋白质磷酸化的理解，我们开发了一种系统地识别蛋白质组装体的磷酸化依赖性的方法。非特异性蛋白磷酸酶处理，尺寸排阻色谱法和质谱法的组合使我们能够确定去除磷酸修饰后蛋白质相互作用的变化。通过这种方法，我们能够鉴定出316种参与磷酸化敏感相互作用的蛋白质。我们恢复了已知的磷酸化依赖性相互作用，如FACT复合物和剪接体，以及确定了新的相互作用，如三肽基肽酶TPP2和超剪接体成分ZRANB2。更一般地说，我们发现磷酸化依赖的相互作用强烈富集RNA结合蛋白，提供了新的见解磷酸化在RNA结合中的作用。通过直接搜索质谱数据中的磷酸化氨基酸残基，我们确定了ZRANB2和FACT复合物亚基SSRP 1上可能的调节磷酸化位点。这项研究提供了一种方法和资源，以获得更好地了解磷酸化的作用，在天然大分子组装。所有质谱数据可通过PRIDE获得（登录号PXD 021422）。
Protein phosphorylation is a key regulatory mechanism involved in nearly every eukaryotic cellular process. Increasingly sensitive mass spectrometry approaches have identified hundreds of thousands of phosphorylation sites but the functions of a vast majority of these sites remain unknown, with fewer than 5% of sites currently assigned a function. To increase our understanding of functional protein phosphorylation we developed an approach for identifying the phosphorylation-dependence of protein assemblies in a systematic manner. A combination of non-specific protein phosphatase treatment, size-exclusion chromatography, and mass spectrometry allowed us to identify changes in protein interactions after the removal of phosphate modifications. With this approach we were able to identify 316 proteins involved in phosphorylation-sensitive interactions. We recovered known phosphorylation-dependent interactors such as the FACT complex and spliceosome, as well as identified novel interactions such as the tripeptidyl peptidase TPP2 and the supraspliceosome component ZRANB2. More generally, we find phosphorylation-dependent interactors to be strongly enriched for RNA-binding proteins, providing new insight into the role of phosphorylation in RNA binding. By searching directly for phosphorylated amino acid residues in mass spectrometry data, we identified the likely regulatory phosphosites on ZRANB2 and FACT complex subunit SSRP1. This study provides both a method and resource for obtaining a better understanding of the role of phosphorylation in native macromolecular assemblies. All mass spectrometry data is available through PRIDE (Accession #PXD021422).
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影响因子：
12.3
作者：
Hart GT;Ramani AK;Marcotte EM
通讯作者：
Marcotte EM
DOI：
10.1073/pnas.1017700108
发表时间：
2011-05-17
影响因子：
11.1
作者：
Cho, Suhyung;Hoang, Amy;Ghosh, Gourisankar
通讯作者：
Ghosh, Gourisankar
影响因子：
11.4
作者：
Landry, Christian R.;Levy, Emmanuel D.;Michnick, Stephen W.
通讯作者：
Michnick, Stephen W.
影响因子：
48
作者：
Huang, Jun X.;Lee, Gihoon;Moellering, Raymond E.
通讯作者：
Moellering, Raymond E.
影响因子：
7.5
作者：
Hall, Frederick L.;Vulliet, P. Richard
通讯作者：
Vulliet, P. Richard