Protein-dimensional Liquid Chromatograph And Applications,

A new type of on-line two-dimensional liquid chromatography(2D-LC) for protein separation system was built by the improvement of high-performance liquid chromatograph.It was assembled to be a multi-functional and automated multi-dimensional liquid chromatography (mD-LC) apparatus for proteomics and it also can be connected to any a liquid chromatograph as an independent auxiliary equipment and named as "ChromatoExpert".On this basis,the function of two-dimensional liquid chromatography using single-column(2DLC-1C) on this apparatus was carried out.Literature review:The development of 2D-LC and mD-LC instruments was introduced and reviewed in detail.The scheme of improvement devices was proposed by the analyzing of the advantages and disadvantages of the reported 2D-LC and mD-LC apparatus.The automated 2D-LC and mD-LC apparatus were summarized briefly.The experimental method was introduced,including apparatus,reagent,as well as the methods of enzyme activity,SDS-polyacrylamide gel electrophoresis and protein determination.An eqipment called as "ChromatoExpert" for prossing fast protein separation on-line 2D-LC by a single column(2D-LC-1C) is firstly constructed.The "ChromatoExpert" has multiple functions as:on-line fraction collection from the first separation mode and quantitatively sample re-injection into the second separation mode,on-line buffer exchange.The collected fraction can be storaged in the equipment at 4℃and also can sterilize in situ with high water steam.By taking the device as an example,the operation principle of the apparatus was discussed profoundly.Using one weak cation exchange(WCX) and hydrophobic functions mixed-mode column on the apparatus,2D-LC-1C separating standard proteins was carried out successfully.Finally,mass and activity recovery were tested,and good results was obtained.Using WCX-HIC mixed mode column on-line 2D-LC-1C separation and purification the bovine pancreas chymotrypsin and trypsin,higher purity of the target proteins,and better mass and activity recovery were obtained.Finally,the prospect of ChromatoExpert for future development was viewed.