Muntel Jan, Fromion Vincent, Goelzer Anne, Maaβ Sandra, Mäder Ulrike, Büttner Knut, Hecker Michael, Becher Dörte
Institute for Microbiology, Ernst Moritz Arndt University Greifswald, D-17487 Greifswald, Germany;
Mol Cell Proteomics. 2014 Apr;13(4):1008-19. doi: 10.1074/mcp.M113.032631. Epub 2014 Jan 31.
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/MS(E)). 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/MS(E)) data has been performed for the first time and should pave the way for future comprehensive quantitative characterization of microorganisms as physiological entities.
在不断发展的系统生物学领域，要真正理解细胞内的代谢和适应网络，对蛋白质浓度的了解至关重要。因此，我们建立了一种工作流程，该流程依赖于长色谱分离和质谱分析，通过同时对所有前体离子进行数据独立的平行碎片化（液相色谱/质谱联用（E））。通过防止对共洗脱的低丰度和高丰度肽的歧视，可实现40%的高平均序列覆盖率，从而鉴定出革兰氏阳性模式生物枯草芽孢杆菌预测的胞质蛋白质组的近一半（>1050种蛋白质）。通过将蛋白质的三种最强肽段的平均质谱信号强度与加标的标准蛋白质消化物的信号强度进行关联，实现了绝对定量。与重标记肽段的比较分析（AQUA方法）表明，仅使用一种标准消化物就足以进行全局定量。定量结果涵盖了近四个数量级，大致范围为每个细胞10到150000个拷贝。为了证明该方法的生物学相关性，我们分析了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。这尤其能够验证已知调控事件对蛋白质水平的调节作用，总体上也为深入了解细菌生理学提供了新的视角。在新发现中，对细胞过程“蛋白质成本”的分析极其重要。基于液相色谱/质谱联用（LC/MS（E））数据中的数据独立平行碎片化，首次对细胞蛋白质浓度进行了如此全面和详细的表征，这应该为未来将微生物作为生理实体进行全面定量表征铺平道路。
