Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS
Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS
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DOI：
10.7554/elife.27574
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发表时间：
2017-10-20
期刊：
影响因子：
7.7
通讯作者：
Lamond, Angus I.
中科院分区：
文献类型：
--
作者：
Ly, Tony;Whigham, Arlene;Lamond, Angus I.
蛋白质丰度和翻译后修饰的时间调节是细胞分裂的关键特征。最近，我们分析了在最小扰动条件下的间期基因表达和蛋白质丰度的变化(Ly等人，2014,2015)。在这里，我们表明，通过使用特定的细胞内免疫标记方案，FACS分离间期和有丝分裂细胞，包括有丝分裂亚相，可以与蛋白质组学分析相结合。使用这种PRIMMUS(细胞内未标记细胞亚群的蛋白质组学分析)方法，我们现在比较了来自非同步生长的人类细胞的间期和有丝分裂部分的蛋白质丰度和磷酸化变化。我们发现了一组在G2期间增加的115个磷酸化位点，称为‘早起’。这一组包括S738在TPX2上的磷酸化，我们表明这对TPX2的功能和有丝分裂进程是重要的。此外，我们使用PRIMMUS首次提供了前期、前期和后期之间蛋白质丰度重塑的蛋白质组学分析。
The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed 'early risers'. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.