title: A fully automated dual-online multifunctional ultrahigh pressure liquid chromatography system for high-throughput proteomics analysis.
abstract: A fully automated dual-online multifunctional ultrahigh pressure liquid chromatography (DO-MULTI-UPLC) system has been developed for high throughput proteome analyses of complex peptide mixtures. The system employs two online solid phase extraction (SPE) columns (150μm inner diameter×3cm), two capillary reverse phase (RP) columns (75μm×100cm) and a strong cation exchange (SCX) column (150μm×15cm) on a single system utilizing one binary pump and one isocratic pump. With the automated operation of six switching valves, the selection of LC experiments between single-dimensional RPLC and online two-dimensional SCX/RPLC were achieved automatically, without manual intervention, while two RPLC columns were used independently and alternatively. By essentially removing the dead time for column equilibration between experiments, in either 1D mode or 2D experimental mode, the current system was demonstrated to increase the experimental throughput by about two folds, while keeping the inter-column reproducibility of peptide elution time in less than 1% of gradient time. The advantageous features of the proposed system were demonstrated by its application to proteome samples of varying complexities.
authors.affiliation: Department of Chemistry, Research Institute for Natural Sciences, Korea University, Seoul 136-701, South Korea. Electronic address: hanhgr@korea.ac.kr.
authors.forename: Hangyeore
authors.initials: H
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Research Institute for Natural Sciences, Korea University, Seoul 136-701, South Korea. Electronic address: qocn19@hanmail.net.
authors.forename: Jung Hwa
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Research Institute for Natural Sciences, Korea University, Seoul 136-701, South Korea. Electronic address: javamng@hotmail.com.
authors.forename: Hokeun
authors.initials: H
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Research Institute for Natural Sciences, Korea University, Seoul 136-701, South Korea. Electronic address: suechin@hanmail.net.
authors.forename: Su-Jin
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Research Institute for Natural Sciences, Korea University, Seoul 136-701, South Korea. Electronic address: bjg0926@korea.ac.kr.
authors.forename: Jingi
authors.initials: J
authors.lastname: Bae
authors.affiliation: National Cancer Center, Goyang 410-769, South Korea. Electronic address: harkkimmd@gmail.com.
authors.forename: Hark Kyun
authors.initials: HK
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Research Institute for Natural Sciences, Korea University, Seoul 136-701, South Korea. Electronic address: sw_lee@korea.ac.kr.
authors.forename: Sang-Won
authors.initials: SW
authors.lastname: Lee
mesh_terms: D057205:Automation, Laboratory / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D009862:Online Systems; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D052616:Solid Phase Extraction
chemical_list: D020543:Proteome
keywords: Bottom-up proteomics; Dual-online LC; Multidimensional separation; Tandem mass spectrometry