凝胶过滤/离子交换全二维液相色谱系统的构建与评价

基于蛋白质的尺寸及带电性质,将凝胶过滤色谱(GFC)与离子交换色谱(IEC)两种分离模式结合,采用双捕集柱接口构建了GFC/2×IEC二维液相色谱(2-D LC)分离系统,同时考虑离子交换色谱分离蛋白质对等电点范围的限制,进一步结合中心切割平行柱的方法实现对蛋白质的全二维分离。为与后续蛋白质在线酶解、多肽分离及质谱鉴定匹配,系统中采用常规柱以保证蛋白质质谱鉴定对样品量的要求,3种常规分离柱分别选用凝胶过滤色谱柱TSK-GEL G3000SW_(XL)(300 mm×7.8 mm,5μm)、强阴离子交换色谱柱Hypersil SAX(100 mm×4.6 mm,10μm)和强阳离子交换色谱柱Hypersil SCX(100 mm×4.6 mm,10μm)。最终以酵母细胞蛋白质提取液为样品,对构建的二维系统加以评价,在总蛋白质浓度13.5 mg/mL、上样体积100μL的条件下,将第一维分离等时间切割17次,并将切割馏分全部导入第二维继续分离,二维系统在148 min内获得的总峰容量达到884。说明所构建的系统可以用于蛋白质的在线全二维分离。

Construction and evaluation of gel filtration/ion exchange comprehensive two-dimensional liquid chromatography system

A two-dimensional liquid chromatography (2-D LC) system was developed with gel filtration chromatography (GFC) and ion exchange chromatography (IEC) based on the sizes and electric properties of proteins. A double-trap-column interface was used in the GFC/2×IEC 2-D LC system. To achieve the comprehensive 2-D separation, heart-cutting parallel columns were used in the second dimension considering the isoelectric point range of proteins. A general proteomic platform was established with protein separation, online digestion, peptide separation and mass identification. A TSK-GEL G3000SW_XL column (300 mm×7.8 mm, 5 μm), a Hypersil SAX column (100 mm×4.6 mm, 10 μm) and a Hypersil SCX column (100 mm×4.6 mm, 10 μm) were used to separate the yeast protein samples. The GFC/2×IEC 2-D LC system was evaluated with the yeast protein samples (100 μL) of concentration of 13.5 mg/mL. First dimensional GFC fractions were cut for 17 times within 148 min and transferred continuously to the second dimension to continue the separation. Under the optimized conditions, the total peak capacity of 2-D LC system reached 884. The results demonstrated that the 2-D LC system can be used for on-line protein separation. The protein separation module has a great prospect in the field of proteomic research.