title: Weak anion and cation exchange mixed-bed microcolumn for protein separation.
abstract: To separate proteins with a wide distribution of pIs under the conditions compatible to online tryptic digestion (with preferable pH=8.0), weak anion and cation exchange chromatography (WAX/WCX) mixed-bed microcolumn has been developed. With a mixture of five proteins with pIs ranging from 4.2 to 11.4, the effect of WAX/WCX ratio on the separation performance was investigated, and an optimum packing ratio of 1:1 w/w was obtained. Moreover, the undesirable hydrophobic interaction between the proteins and the stationary phase was suppressed with 10% ACN v/v added in the mobile phases. Under the optimized conditions compatible to tryptic digestion, basic and acidic proteins were resolved simultaneously, with RSDs of relative retention time on six columns less than 6%, indicating the good resolution and packing reproducibility. Furthermore, one RPLC fraction of proteins extracted from rat middle brain and the whole protein mixture extracted from rat liver were analyzed, respectively. The results demonstrated better separation performance on WAX/WCX microcolumns than that on both weak anion exchange chromatography and weak cation exchange chromatography at pH ∼8. We anticipate that WAX/WCX microcolumns are promising for the integration of protein separation and tryptic digestion aiming at high-throughput proteome study.
authors.affiliation: Key Lab of Separation Science for Analytical Chemistry, National Chromatographic R.&A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, PR China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Hou
authors.forename: Huiming
authors.initials: H
authors.lastname: Yuan
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Qiao
authors.forename: Jinxiang
authors.initials: J
authors.lastname: Liu
authors.forename: Yichu
authors.initials: Y
authors.lastname: Shan
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000838:Anions / Q000737:chemistry*; D001923:Brain Chemistry; D002412:Cations / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000295:instrumentation* / Q000379:methods; D007526:Isoelectric Point; D008099:Liver / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*; D051381:Rats
chemical_list: D000838:Anions; D002412:Cations; D011506:Proteins