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

doi:10.3724/SP.J.1123.2016.12006

Abstract

Abstract:

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.

Key words: comprehensive two-dimensional liquid chromatography (2-D LC), gel filtration chromatography (GFC), ion exchange chromatography (IEC), protein, strong anion exchange (SAX), strong cation exchange (SCX), yeast

CLC Number:

O658

ZHANG Zheng, TANG Tao, YANG Sandong, SUN Yuanshe, LI Tong, ZHANG Weibing. Construction and evaluation of gel filtration/ion exchange comprehensive two-dimensional liquid chromatography system[J]. Chinese Journal of Chromatography, 2017, 35(5): 526-532.

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