title: Integrated platform for proteome profiling with combination of microreversed phase based protein and peptide separation via online solvent exchange and protein digestion.
abstract: An online integrated platform for proteome profiling was established, with the combination of protein separation by microreversed phase liquid chromatography (μRPLC), online acetonitrile (ACN) removal, and pH adjustment by a hollow fiber membrane interface (HFMI), online digestion by an immobilized enzymatic microreactor (IMER), as well as peptide separation and proteins identification by μRPLC or nano-RPLC-electrospray ionization tandem mass spectrometry (μRPLC-ESI-MS/MS). To evaluate the performance of such a platform, a three-protein mixture with mass ranging from 5 to 500 ng was analyzed automatically. Compared to the offline counterpart, although similar protein sequence coverages were obtained by the integrated platform, the signal intensity of total ion chromatogram was improved by almost 4 times. In addition, such an integrated platform was further applied for the analysis of extracted proteins from rat brain. Compared to the results obtained by offline counterpart and traditional MudPIT approach under similar conditions, by the integrated platform, the identified protein group number was comparable, but the analysis time was shortened to less than half of that taken by the traditional approaches. All these results demonstrated that our developed integrated platform might offer a promising tool for high-throughput and large-scale profiling of proteomes.
authors.affiliation: National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Huiming
authors.initials: H
authors.lastname: Yuan
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Simin
authors.initials: S
authors.lastname: Xia
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Zhang
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000818:Animals; D057205:Automation, Laboratory; D001923:Brain Chemistry; D002417:Cattle; D005591:Chemical Fractionation / Q000379:methods; D056148:Chromatography, Reverse-Phase; D057166:High-Throughput Screening Assays; D006736:Horses; D006863:Hydrogen-Ion Concentration; D059748:Proteolysis; D020543:Proteome / Q000032:analysis*; D051381:Rats; D012997:Solvents; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D020543:Proteome; D012997:Solvents; D014357:Trypsin; C032159:acetonitrile