title: Serially coupled microcolumn reversed phase liquid chromatography for shotgun proteomic analysis.
abstract: Microcolumn RPLC (microRPLC) is one of the optimum separation modes for shotgun proteomic analysis. To identify as many proteins as possible by MS/MS, the improvement on separation efficiency and peak capacity of muRPLC is indispensable. Although the increase in column length is one of the effective solutions, the preparation of a long microcolumn is rather difficult due to the high backpressure generated during the packing procedure. In our recent work, through connecting microcolumns of 5, 10, and 15 cm length via unions with minimal dead volume, long microcolumns with length up to 30 cm were obtained, with which 318 proteins were identified from proteins extracted from Escherichia coli by microRPLC-ESI MS/MS, and similar distributions of M(w) and pI were found with single and various coupled microcolumns. Furthermore, by using MS/MS with improved sensitivity, with such a serially coupled 30 cm long microcolumn, 1692 proteins were identified within 7 h from rat brain tissue, with false positive rate (FPR) <1%. All these results demonstrated that serially couple microcolumns might be of great promising to improve the separation capacity of microRPLC in shotgun proteomic analysis.
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
authors.forename: Dingyin
authors.initials: D
authors.lastname: Tao
authors.forename: Guijie
authors.initials: G
authors.lastname: Zhu
authors.forename: Liangliang
authors.initials: L
authors.lastname: Sun
authors.forename: Junfeng
authors.initials: J
authors.lastname: Ma
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Weibing
authors.initials: W
authors.lastname: Zhang
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis*; D008832:Microchemistry / Q000379:methods*; D011312:Pressure; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D029968:Escherichia coli Proteins