Microwave-assisted protein preparation and enzymatic digestion in proteomics.
The combinations of gel electrophoresis or LC and mass spectrometry are two popular approaches for large scale protein identification. However, the throughput of both approaches is limited by the speed of the protein digestion process. Present research into fast protein enzymatic digestion has been focused mainly on known proteins, and it is unclear whether these results can be extrapolated to complex protein mixtures. In this study microwave technology was used to develop a fast protein preparation and enzymatic digestion method for protein mixtures. The protein mixtures in solution or in gel were prepared and digested by microwave-assisted protein enzymatic digestion, which rapidly produces peptide fragments. The peptide fragments were further analyzed by capillary LC and ESI-ion trap-MS or MALDI-TOF-MS. The technique was optimized using bovine serum albumin and then applied to human urinary proteins and yeast lysate. The method enabled preparation and digestion of protein mixtures in solution (human urinary proteins) or in gel (yeast lysate) in 6 or 25 min, respectively. Equivalent (in-solution) or better (in-gel) digestion efficiency was obtained using microwave-assisted protein enzymatic digestion compared with the standard overnight digestion method. This new application of microwave technology to protein mixture preparation and enzymatic digestion will hasten the application of proteomic techniques to biological and clinical research.
Sun
Wei
W
Proteomics Research Center and National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Peking Union Medical College/Chinese Academy of Medical Sciences, Beijing 100005, China. sunwei1018@hotmail.com
Gao
Shijuan
S
Wang
Linjie
L
Chen
Yong
Y
Wu
Shuzhen
S
Wang
Xiaorong
X
Zheng
Dexian
D
Gao
Youhe
Y
D002853:Chromatography, Liquid; D006801:Humans; D008872:Microwaves; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011506:Proteins