title: imFASP: An integrated approach combining in-situ filter-aided sample pretreatment with microwave-assisted protein digestion for fast and efficient proteome sample preparation.
abstract: An integrated sample preparation method, termed "imFASP", which combined in-situ filter-aided sample pretreatment and microwave-assisted trypsin digestion, was developed for preparation of microgram and even nanogram amounts of complex protein samples with high efficiency in 1 h. For imFASP method, proteins dissolved in 8 M urea were loaded onto a filter device with molecular weight cut off (MWCO) as 10 kDa, followed by in-situ protein preconcentration, denaturation, reduction, alkylation, and microwave-assisted tryptic digestion. Compared with traditional in-solution sample preparation method, imFASP method generated more protein and peptide identifications (IDs) from preparation of 45 μg Escherichia coli protein sample due to the higher efficiency, and the sample preparation throughput was significantly improved by 14 times (1 h vs. 15 h). More importantly, when the starting amounts of E. coli cell lysate decreased to nanogram level (50-500 ng), the protein and peptide identified by imFASP method were improved at least 30% and 44%, compared with traditional in-solution preparation method, suggesting dramatically higher peptide recovery of imFASP method for trace amounts of complex proteome samples. All these results demonstrate that the imFASP method developed here is of high potential for high efficient and high throughput preparation of trace amounts of complex proteome samples.
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian, 116023, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian, 116023, China; Graduate School of Chinese Academy of Sciences, Beijing, 100039, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Fang
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian, 116023, China; Graduate School of Chinese Academy of Sciences, Beijing, 100039, China.
authors.forename: Ci
authors.initials: C
authors.lastname: Wu
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian, 116023, China; Graduate School of Chinese Academy of Sciences, Beijing, 100039, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian, 116023, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian, 116023, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian, 116023, China. Electronic address: lihuazhang@dicp.ac.cn.
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian, 116023, China.
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D002853:Chromatography, Liquid; D008872:Microwaves; D011506:Proteins / Q000737:chemistry*; D020543:Proteome; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D011506:Proteins; D020543:Proteome
keywords: Filter; In-situ; Microwave; Sample preparation; Trace proteome analysis