Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS: Preparation of more than 4000 native protein maps

Soluble proteins of human bronchial smooth muscle cells (HBSMC) were separated by nondenaturing micro 2DE and a 30 mm X 40 mm area of the CBB-stained slab gel (1.0 mm thick) was cut into 1.1 mm X 1.1 mm squares, then the proteins in the 972 gel pieces (squares) were applied to quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data [Jin, et al. Electrophoresis 2014, 35, 2055-2064, PRIDE Project PXD000852]. Totally 4323 proteins were identified in successfully analyzed 967 squares and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The quantity of the proteins distributed from 3.6 % to 1 X 10-5 % of the total protein quantity in the grid area. Each protein map was characterized by many features, such as the position of quantity peak square, number of detected squares, degree of concentration (focused or dispersed), etc. About 4 percent of the proteins were detected in 100 or more squares, suggesting that they might be ubiquitous and interacting with other proteins. In contrast, many proteins showed more concentrated quantity distribution and the quantity peak positions of 565 proteins with defined degree of concentration were summarized into a quantity peak map. These results for the first time visualized the distribution patterns of cellular proteins on a nondenaturing 2DE gel, which would further provide information on their interactions.

本研究采用非变性微型双向凝胶电泳（nondenaturing micro 2DE）分离人类支气管平滑肌细胞（HBSMC）的可溶性蛋白质，将考马斯亮蓝（CBB）染色的1.0 mm厚平板凝胶上30 mm×40 mm的区域切割为1.1 mm×1.1 mm的小块，随后对972个凝胶块中的蛋白质进行定量液相色谱-串联质谱（quantitative LC-MS/MS）分析。
本研究采用凝胶网格切割法实现三大目标：(i) 确保该区域内蛋白质的全面分析；(ii) 标准化液相色谱-串联质谱的分析条件；(iii) 基于定量数据重构蛋白质分布模式[Jin等，Electrophoresis 2014, 35, 2055-2064, PRIDE项目PXD000852]。
在成功完成分析的967个凝胶块中，共鉴定出4323种蛋白质，每种蛋白质的含量分布被重构为颜色密度模式（即天然蛋白质图谱）。该网格区域内蛋白质的含量占总蛋白量的比例范围为3.6%至1×10^-5%。
每张蛋白质图谱均具备多项特征，例如含量峰值所在凝胶块的位置、被检测到的凝胶块数量、聚集程度（集中或分散）等。约4%的蛋白质在100个及以上的凝胶块中被检出，提示这类蛋白质可能广泛存在并与其他蛋白质存在相互作用。
与之相反，多数蛋白质的含量分布更为集中；研究人员将565个具有明确聚集程度的蛋白质的含量峰值位置整合为一张含量峰值图谱。本研究首次实现了非变性双向凝胶电泳平板上细胞蛋白质分布模式的可视化，可为后续探究蛋白质相互作用提供关键信息。