Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes
Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes
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DOI：
10.1002/pmic.200500352
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发表时间：
2006-04-01
期刊：
影响因子：
3.4
通讯作者：
Séror, SJ
中科院分区：
文献类型：
--
作者：
Lévine, A;Vannier, F;Séror, SJ
丝氨酸/苏氨酸/酪氨酸（Ser/Thr/Tyr）残基上磷酸化的蛋白质的生理作用或相应激酶和磷酸酶的身份在细菌中通常知之甚少。作为第一步，在分析这种磷酸化的重要性，我们试图建立的性质的丝氨酸/苏氨酸/酪氨酸磷酸化蛋白质组在枯草芽孢杆菌，使用在体内标记与[P-32]-正磷酸盐，一个单位的pH 2-DE，结合MS。2-D曲线在pH 4-7范围内包含至少80个清晰标记的斑点。通过MS分析了46个点（在大多数情况下通过LC-MS/MS确认），共鉴定出29种不同的蛋白质，其中19种首次鉴定为细菌磷蛋白。这些磷蛋白参与多种细胞过程，包括碳和能量代谢、运输、应激和发育。显着变化的配置文件得到的结果冷，热或渗透压休克，表明，在静止期细胞，磷酸化蛋白质组是动态的。初步比较研究表明，至少有25个[P-32]标记的斑点也被Pro-Q Diamond染色，显然还有6个额外的磷蛋白被Pro-Q唯一检测到。
The physiological role of proteins phosphorylated on serine/threonine/tyrosine (Ser/Thr/Tyr) residues or the identity of the corresponding kinases and phosphatases is generally poorly understood in bacteria. As a first step in analysing the importance of such phospborylation, we sought to establish the nature of the Ser/Thr/Tyr phosphoproteome in Bacillus subtilis, using in vivo labelling with [P-32]-orthophosphate, one-unit pH 2-DE, combined with MS. Highly reproducible 2-D profiles of phosphoproteins were obtained with early stationary-phase cells. The 2-D profiles contained at least 80 clearly labelled spots in the pH range 4-7. Forty-six spots were analysed by MS (confirmed in most cases by LC-MS/MS), identifying a total of 29 different proteins, with 19 identified for the first time as bacterial phosphoproteins. These phosphoproteins are implicated in a wide variety of cellular processes, including carbon and energy metabolism, transport, stress and development. Significant changes to the profiles were obtained as a result of cold, heat or osmotic shock, demonstrating that, in stationary-phase cells, the phosphoproteome is dynamic. An initial comparafive study indicated that at least 25 [P-32]-labelled spots were also stained by Pro-Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro-Q.