Construction of a high-performance system to collect protein information by the combination of non-denaturing two-dimensional electrophoresis and mass spectrometry

以人血浆为复杂蛋白质混合物的模型，结合非变性微二维凝胶电泳（我们将这种类型的2-DE称为Type-I micro 2-DE）和蛋白质的质谱归属，构建了一个高性能的收集完整蛋白质和蛋白质复合物信息的系统。通过基质辅助激光解吸电离质谱（MALDI MS）和肽质量指纹图谱技术，对Ⅰ型2-DE凝胶上所有染色点的多肽进行鉴定，收集完整蛋白质和蛋白质复合物的信息。其次，使用与非变性2-DE相同的非变性等电聚焦条件，在十二烷基硫酸钠（SDS）存在下进行二维电泳（我们将这种类型的2-DE称为II型微2-DE），并再次通过质谱法对所有染色点中的多肽进行归属。多肽定位结果之间的比较提供了蛋白质亚基之间和/或不同蛋白质之间的非共价相互作用的信息，这对于发挥蛋白质或蛋白质复合物的特定功能至关重要。第三，我们建立了一种从Ⅰ型微型双向凝胶上的每个染色点提取蛋白质的方法，并利用SDS凝胶电泳（第三维电泳）对每个泳道上的染色条带进行分析，从而揭示了染色点所包含的组成多肽，从而提供了蛋白质-蛋白质相互作用的详细信息。结合这三种方法得到的结果，以及微型2-DE系统的高效率，我们可以收集人血浆中蛋白质和蛋白质复合物的信息。

A high-performance system to collect information on intact proteins and protein complexes was constructed combining non-denaturing micro two-dimensional gel electrophoresis (we named this type of 2-DE as Type-I micro 2-DE) and mass spectrometric assignment of the proteins, using human plasma as a model of complex protein mixtures. Information on intact proteins and protein complexes was firstly collected by assigning polypeptides in all the stained spots on the Type-I 2-DE gel by matrix-assisted laser desorption-ioninzation mass spectrometry (MALDI MS) and peptide mass fingerprinting. Secondly, using the same conditions of non-denaturing isoelectric focusing as the non-denaturing 2-DE, second dimension electrophoresis was run in the presence of sodium dodecyl sulfate (SDS) (we named this type of 2-DE as Type-II micro 2-DE) and again the polypeptides in all the stained spots were assigned by mass spectrometry. The comparisons between the results of polypeptide assignment provided information on non-covalent interactions between protein subunits and/or between different proteins, which should be crucial to exert the specific functions of the proteins or protein complexes. Thirdly, we developed a method to extract proteins from each stained spot on the Type-I micro 2-DE gel and analyze them using SDS-gel electrophoresis (third-dimension electrophoresis).The analysis of the stained bands on each lane of the SDS gel revealed the constituent polypeptides included in the stained spot, thus providing detailed information on the protein-protein interactions. Combining the results obtained by these three methods, together with the high-efficiency of the micro 2-DE system, we could collect information on proteins and protein complexes in human plasma.