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

点击翻译按钮获取中文摘要

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.