Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MSE)*
Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MSE)*
复制标题
DOI：
10.1074/mcp.m113.032631
复制
发表时间：
2014-04-01
影响因子：
7
通讯作者：
Becher, Doerte
中科院分区：
文献类型：
--
作者：
Muntel, Jan;Fromion, Vincent;Becher, Doerte
在不断发展的系统生物学领域，非常需要蛋白质浓度的知识才能真正了解细胞内的代谢和适应网络。因此，我们建立了一个依赖于长色谱分离和质谱分析的工作流程，通过数据独立、同时对所有母离子进行并行裂解 (LC/MSE)。通过防止共洗脱低丰度肽和高丰度肽的区分，可以实现 40% 的高平均序列覆盖率，从而鉴定出几乎一半的革兰氏阳性模型生物枯草芽孢杆菌的预测胞质蛋白质组（> 1,050 个蛋白质）。通过将蛋白质的三种最强肽的平均 MS 信号强度与加标标准蛋白质消化物的信号强度相关联来实现绝对定量。与重标记肽的比较分析（AQUA 方法）表明，仅使用一种标准消化物就足以进行全局定量。定量结果涵盖几乎四个数量级，范围大致为每个细胞 10 到 150,000 个拷贝。为了证明该方法的生物学相关性，对在需要氨基酸合成或氨基酸降解的条件下生长的枯草芽孢杆菌细胞的选定生理方面进行了分析。这尤其可以验证已知调控事件对蛋白质水平的调整，并且总体上可以对细菌生理学有新的见解。在新发现中，细胞过程的“蛋白质成本”分析极其重要。这种基于数据独立的、液相色谱/质谱（LC/MSE）数据中的平行碎片的细胞蛋白浓度的全面和详细的表征是首次进行的，并且应该为未来作为生理实体的微生物的全面定量表征铺平道路。
In the growing field of systems biology, the knowledge of protein concentrations is highly required to truly understand metabolic and adaptational networks within the cells. Therefore we established a workflow relying on long chromatographic separation and mass spectrometric analysis by data independent, parallel fragmentation of all precursor ions at the same time (LC/MSE). By prevention of discrimination of co-eluting low and high abundant peptides a high average sequence coverage of 40% could be achieved, resulting in identification of almost half of the predicted cytosolic proteome of the Gram-positive model organism Bacillus subtilis (> 1,050 proteins). Absolute quantification was achieved by correlation of average MS signal intensities of the three most intense peptides of a protein to the signal intensity of a spiked standard protein digest. Comparative analysis with heavily labeled peptides (AQUA approach) showed the use of only one standard digest is sufficient for global quantification.The quantification results covered almost four orders of magnitude, ranging roughly from 10 to 150,000 copies per cell. To prove this method for its biological relevance selected physiological aspects of B. subtilis cells grown under conditions requiring either amino acid synthesis or alternatively amino acid degradation were analyzed. This allowed both in particular the validation of the adjustment of protein levels by known regulatory events and in general a perspective of new insights into bacterial physiology. Within new findings the analysis of "protein costs" of cellular processes is extremely important. Such a comprehensive and detailed characterization of cellular protein concentrations based on data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MSE) data has been performed for the first time and should pave the way for future comprehensive quantitative characterization of microorganisms as physiological entities.