Coupling the immobilized trypsin microreactor of monolithic capillary with μRPLC-MS/MS for shotgun proteome analysis

被引:76
作者
Feng, S
Ye, ML
Jiang, XG
Jin, WH
Zou, HF [1 ]
机构
[1] Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, Dalian 116023, Peoples R China
[2] Xinjiang Univ, Coll Chem & Chem Engn, Xinjiang 830046, Peoples R China
关键词
immobilized trypsin; microreactor; monolithic matrix; shotgun proteomics; mu RPLC-MS/MS;
D O I
10.1021/pr0502727
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A nanoliter trypsin-based monolithic microreactor coupled with mu RPLC-MS/MS was reported for shotgun proteome analysis. The proteins were rapidly digested by the microreactor, and the resulting protein digests were directly loaded onto a mu RPLC column for separation followed with detection of the eluted peptides by tandem mass spectrometer. The digestion efficiency and stability of the microreactor was demonstrated by using bovine serum albumin as a model protein. When compared with an incubation time of more than 10 h by free trypsin in the conventional digestion approach, protein mixtures can be digested by the microreactor in several minutes. This system was applied to the analysis of the total cell lysate of Saccharomyces cerevisiae. After a Sequest database search, a total of 1578 unique peptides corresponding to 541 proteins were identified when 590 ng yeast protein was digested by the microreactor with an incubation time of only 1 min.
引用
收藏
页码:422 / 428
页数:7
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