Coupling the immobilized trypsin microreactor of monolithic capillary with μRPLC-MS/MS for shotgun proteome analysis
Coupling the immobilized trypsin microreactor of monolithic capillary with μRPLC-MS/MS for shotgun proteome analysis
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DOI：
10.1021/pr0502727
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发表时间：
2006-02-01
影响因子：
4.4
通讯作者：
Zou, HF
中科院分区：
文献类型：
--
作者：
Feng, S;Ye, ML;Zou, HF
报道了一种基于纳升胰蛋白酶的整体式微反应器与μ RPLC-MS/MS联用进行鸟枪法蛋白质组分析。通过微反应器快速消化蛋白质，并将所得蛋白质酶直接上样到μ RPLC柱上进行分离，然后通过串联质谱仪检测洗脱的肽。以牛血清白蛋白为模型蛋白，考察了微反应器的消化效率和稳定性。与传统消化方法中游离胰蛋白酶超过10 h的孵育时间相比，微反应器可以在几分钟内消化蛋白质混合物。将该系统应用于酿酒酵母总细胞裂解液的分析。在Sequest数据库搜索后，当590 ng酵母蛋白被微反应器消化，孵育时间仅为1 min时，共鉴定出对应于541种蛋白的1578种独特肽。
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.