Collins Mark O, Yu Lu, Campuzano Iain, Grant Seth G N, Choudhary Jyoti S
Proteomic Mass Spectrometry, The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB101SA, United Kingdom.
Mol Cell Proteomics. 2008 Jul;7(7):1331-48. doi: 10.1074/mcp.M700564-MCP200. Epub 2008 Apr 3.
We analyzed the mouse forebrain cytosolic phosphoproteome using sequential (protein and peptide) IMAC purifications, enzymatic dephosphorylation, and targeted tandem mass spectrometry analysis strategies. In total, using complementary phosphoenrichment and LC-MS/MS strategies, 512 phosphorylation sites on 540 non-redundant phosphopeptides from 162 cytosolic phosphoproteins were characterized. Analysis of protein domains and amino acid sequence composition of this data set of cytosolic phosphoproteins revealed that it is significantly enriched in intrinsic sequence disorder, and this enrichment is associated with both cellular location and phosphorylation status. The majority of phosphorylation sites found by MS were located outside of structural protein domains (97%) but were mostly located in regions of intrinsic sequence disorder (86%). 368 phosphorylation sites were located in long regions of disorder (over 40 amino acids long), and 94% of proteins contained at least one such long region of disorder. In addition, we found that 58 phosphorylation sites in this data set occur in 14-3-3 binding consensus motifs, linear motifs that are associated with unstructured regions in proteins. These results demonstrate that in this data set protein phosphorylation is significantly depleted in protein domains and significantly enriched in disordered protein sequences and that enrichment of intrinsic sequence disorder may be a common feature of phosphoproteomes. This supports the hypothesis that disordered regions in proteins allow kinases, phosphatases, and phosphorylation-dependent binding proteins to gain access to target sequences to regulate local protein conformation and activity.
我们使用连续（蛋白质和肽）IMAC纯化、酶促去磷酸化和靶向串联质谱分析策略，分析了小鼠前脑胞质磷酸化蛋白质组。总共，使用互补的磷酸富集和LC-MS/MS策略，对来自162种胞质磷酸化蛋白质的540种非冗余磷酸肽上的512个磷酸化位点进行了表征。对该胞质磷酸化蛋白质数据集的蛋白质结构域和氨基酸序列组成分析表明，其在内在序列无序方面显著富集，并且这种富集与细胞定位和磷酸化状态均相关。通过质谱发现的大多数磷酸化位点位于结构蛋白质结构域之外（97%），但大多位于内在序列无序区域（86%）。368个磷酸化位点位于长的无序区域（超过40个氨基酸长），并且94%的蛋白质包含至少一个这样的长无序区域。此外，我们发现该数据集中有58个磷酸化位点出现在14-3-3结合共有基序中，这些线性基序与蛋白质中的非结构化区域相关。这些结果表明，在该数据集中，蛋白质磷酸化在蛋白质结构域中显著减少，而在无序蛋白质序列中显著富集，并且内在序列无序的富集可能是磷酸化蛋白质组的一个共同特征。这支持了这样的假设，即蛋白质中的无序区域使激酶、磷酸酶和磷酸化依赖性结合蛋白能够接近靶序列，以调节局部蛋白质构象和活性。
