title: Integrated system for extraction, purification, and digestion of membrane proteins.
abstract: An integrated system was developed for directly processing living cells into peptides of membrane proteins. Living cells were directly injected into the system and cracked in a capillary column by ultrasonic treatment. Owing to hydrophilicity for broken pieces of the cell membrane, the obtained membranes were retained in a well-designed bi-filter. While cytoplasm proteins were eluted from the bi-filter, the membranes were dissolved and protein released by flushing 4% SDS buffer through the bi-filter. The membrane proteins were subsequently transferred into a micro-reactor and covalently bound in the reactor for purification and digestion. As the system greatly simplified the whole pretreatment processes and minimized both sample loss and contamination, it could be used to analyze the membrane proteome samples of thousand-cell-scales with acceptable reliability and stability. We totally identified 1348 proteins from 5000 HepG2 cells, 615 of which were annotated as membrane proteins. In contrast, with conventional method, only 233 membrane proteins were identified. It is adequately demonstrated that the integrated system shows promising practicability for the membrane proteome analysis of small amount of cells.
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China.
authors.forename: Yiying
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China.
authors.forename: Guoquan
authors.initials: G
authors.lastname: Yan
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China.
authors.forename: Mingxia
authors.initials: M
authors.lastname: Gao
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China.
authors.forename: Chunhui
authors.initials: C
authors.lastname: Deng
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China. xmzhang@fudan.edu.cn.
authors.forename: Xiangmin
authors.initials: X
authors.lastname: Zhang
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D056945:Hep G2 Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D020543:Proteome; D015203:Reproducibility of Results; D018511:Systems Integration
chemical_list: D008565:Membrane Proteins; D020543:Proteome
keywords: Bi-filter; Integrated system; Membrane proteins; Micro-reactor