title: Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn.
abstract: To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns.
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, P. R. China.
authors.forename: Dingyin
authors.initials: D
authors.lastname: Tao
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.forename: Guijie
authors.initials: G
authors.lastname: Zhu
authors.forename: Yu
authors.initials: Y
authors.lastname: Liang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods*; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D020543:Proteome / Q000032:analysis*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation* / Q000379:methods*
chemical_list: D011506:Proteins; D020543:Proteome