title: Development of continuous microwave-assisted protein digestion with immobilized enzyme.
abstract: In this study, an easy and efficiency protein digestion method called continuous microwave-assisted protein digestion (cMAED) with immobilized enzyme was developed and applied for proteome analysis by LC-MS(n). Continuous microwave power outputting was specially designed and applied. Trypsin and bromelain were immobilized onto magnetic micropheres. To evaluate the method of cMAED, bovine serum albumin (BSA) and protein extracted from ginkgo nuts were used as model and real protein sample to verify the digestion efficiency of cMAED. Several conditions including continuous microwave power, the ratio of immobilized trypsin/BSA were optimized according to the analysis of peptide fragments by Tricine SDS-PAGE and LC-MS(n). Subsequently, the ginkgo protein was digested with the protocols of cMAED, MAED and conventional heating enzymatic digestion (HED) respectively and the LC-MS(n) profiles of the hydrolysate was compared. Results showed that cMAED combined with immobilized enzyme was a fast and efficient digestion method for protein digestion and microwave power tentatively affected the peptide producing. The cMAED method will be expanded for large-scale preparation of bioactive peptides and peptide analysis in biological and clinical research.
authors: College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China.
authors: Zhengyi
authors: Z
authors: Chen
authors: Shenzhen Academy of Metrology and Quality Inspection, Shenzhen 518131, China.
authors: Yongle
authors: Y
authors: Li
authors: Department of Chemistry, Hong Kong Baptist University, Hong Kong, China.
authors: Shuhai
authors: S
authors: Lin
authors: College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China.
authors: Meiping
authors: M
authors: Wei
authors: College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China.
authors: Fuyou
authors: F
authors: Du
authors: College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China. Electronic address: guihuaruan@hotmail.com.
authors: Guihua
authors: G
authors: Ruan
mesh_terms: D000818:Animals; D001963:Bromelains / Q000378:metabolism*; D002417:Cattle; D002853:Chromatography, Liquid; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004867:Equipment Design; D020441:Ginkgo biloba / Q000737:chemistry; D013058:Mass Spectrometry; D008872:Microwaves; D008958:Models, Molecular; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D010940:Plant Proteins / Q000032:analysis / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000295:instrumentation*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000378:metabolism; D013552:Swine; D014357:Trypsin / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D010455:Peptides; D010940:Plant Proteins; D012710:Serum Albumin, Bovine; D001963:Bromelains; D014357:Trypsin
keywords: Continuous microwave-assisted enzymatic digestion; Immobilized enzyme; Peptide analysis; Proteomics