Quantitative phosphoproteome analysis of a mouse liver cell line reveals specificity of phosphatase inhibitors
Quantitative phosphoproteome analysis of a mouse liver cell line reveals specificity of phosphatase inhibitors
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DOI：
10.1002/pmic.200800105
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发表时间：
2008-11-01
期刊：
影响因子：
3.4
通讯作者：
Mann, Matthias
中科院分区：
文献类型：
--
作者：
Pan, Cuiping;Gnad, Florian;Mann, Matthias
肝脏是涉及生理和疾病的许多方面的中心器官。肝细胞（主要的肝细胞类型）的信号传导特性在代谢疾病和再生中具有特殊的意义。出于这个原因，我们研究了磷酸化蛋白质组的小鼠肝细胞系Hepa 1 -6的稳定同位素标记的氨基酸在细胞培养（SILAC）和高分辨率MS。使用严格的统计评价标准，我们获得了5433磷酸化位点1808蛋白。磷酸化蛋白质组包括所有主要的蛋白质类别，包括大量的转录因子。我们比较了对照和用S1 LAC处理的磷酸酶抑制剂处理的细胞。这使得能够通过测序更丰富的抑制剂诱导形式的肽，同时仍然观察内源性形式，来容易地鉴定体内磷酸化位点。我们采用过钒酸盐的混合物阻断蛋白酪氨酸磷酸酶（PTP）和calyculin A和溴氰菊酯阻断丝氨酸/苏氨酸磷酸酶的活动。有趣的是，这些常用的抑制剂在标准浓度下仅影响28%的磷酸肽至少两倍。未受影响的位点可能是未被有效抑制的磷酸酶的底物，具有缓慢的动力学或在正常生长的细胞中几乎是化学计量的位点。最后，我们设计了一种三重标记策略，包括对照细胞，刺激的细胞，磷酸酶处理的细胞，以获得磷酸化占用的上限。
The liver is a central organ involved in many aspects of physiology and disease. Signaling properties of hepatocytes, the main liver cell type, are of special interest in metabolic diseases and in regeneration. For this reason we investigated the phosphoproteome of the mouse liver cell line Hepa1-6 by stable isotope labeling by amino acids in cell culture (SILAC) and high resolution MS. Using stringent statistical evaluation criteria, we obtained 5433 phosphorylation sites on 1808 proteins. The phosphoproteome encompasses all major protein classes, including a large number of transcription factors. We compared control and phosphatase inhibitor treated cells by S I LAC. This enabled ready identification of in vivo phosphorylation sites by sequencing the more abundant, inhibitor induced version of the peptide while still observing the endogenous version. We employed a mixture of pervanadate for blocking protein tyrosine phosphatases (PTPs) and calyculin A and deltamethrin for blocking the activities of serine/threonine phosphatases. Interestingly, these commonly used inhibitors in standard concentrations affected only 28% of the phosphopeptides by at least two-fold. The unaffected sites may be substrates of phosphatases that are not efficiently inhibited, have slow kinetic or sites that are almost stoichiometric in normally growing cells. Finally, we devised a triple labeling strategy comprising control cells, stimulated cells, and phosphatase treated cells to derive an upper bound on phosphorylation occupancy.