Rapid, highly efficient extraction and purification of membrane proteins using a microfluidic continuous-flow based aqueous two-phase system.
Membrane proteins play essential roles in regulating various fundamental cellular functions. To investigate membrane proteins, extraction and purification are usually prerequisite steps. Here, we demonstrated a microfluidic aqueous PEG/detergent two-phase system for the purification of membrane proteins from crude cell extract, which replaced the conventional discontinuous agitation method with continuous extraction in laminar flows, resulting in significantly increased extraction speed and efficiency. To evaluate this system, different separation and detection methods were used to identify the purified proteins, such as capillary electrophoresis, SDS-PAGE and nano-HPLC-MS/MS. Swiss-Prot database with Mascot search engine was used to search for membrane proteins from random selected bands of SDS-PAGE. Results indicated that efficient purification of membrane proteins can be achieved within 5-7s and approximately 90% of the purified proteins were membrane proteins (the highest extraction efficiency reported up to date), including membrane-associated proteins and integral membrane proteins with multiple transmembrane domains. Compared to conventional approaches, this new method had advantages of greater specific surface area, minimal emulsification, reduced sample consumption and analysis time. We expect the developed method to be potentially useful in membrane protein purifications, facilitating the investigation of membrane proteomics.
Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics & Molecular Imaging Key Laboratory, Department of Systems Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Rui
R
Hu
Xiaojun
X
Feng
Pu
P
Chen
Meng
M
Fu
Hong
H
Chen
Lin
L
Guo
Bi-Feng
BF
Liu
D002457:Cell Extracts / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D030562:Databases, Protein; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D004867:Equipment Design; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
D002457:Cell Extracts; D008565:Membrane Proteins