title: Nano-flow multidimensional liquid chromatography platform integrated with combination of protein and peptide separation for proteome analysis.
abstract: An integrated multidimensional nano-flow liquid chromatography platform with the combination of protein and peptide separation via online digestion by an immobilized enzymatic reactor was established, and successfully applied for proteome analysis. By this platform, proteins were first separated by a weak anion and weak cation mixed-bed microcolumn under a series of salt steps, online digested by a trypsin immobilized enzymatic reactor, digests trapped and desalted by a C18 precolumn, separated by nano-reversed phase liquid chromatography, and finally identified by electrospray ionization-MS/MS. To evaluate the performance of such a platform, Escherichia coli whole cell lysate proteins were analyzed. Compared with the results obtained by shotgun approach, the identified protein number was increased by 6%, with the consumed time decreased from 38 to 14 h. We also compared with integrate platform based on micro-HPLC, and the required sample amount was decreased to 8 μg. These results demonstrated that such an integrated approach would be an attractive alternative to commonly applied approaches for proteome research.
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: Simin
authors.initials: S
authors.lastname: Xia
authors.forename: Dingyin
authors.initials: D
authors.lastname: Tao
authors.forename: Huiming
authors.initials: H
authors.lastname: Yuan
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhou
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: D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis / Q000302:isolation & purification; D010455:Peptides / Q000032:analysis* / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; D011506:Proteins; D020543:Proteome