An ultra-fast and highly efficient multiple proteases digestion strategy using graphene-oxide-based immobilized protease reagents

查看全文 作　　者：BAI [1,2,3]HaiHong;PAN [1,2,3]YiTing;REN [2,3]XiaoJun;HAO [2,3]FeiRan;DENG [2,3]ShanShan;FAN [2,3]Chao;YAN [2,3]Hui;SHEN [2,3]BingQuan;MA [2,3]Lin;TIAN [2,3]Fang;PENG [2,3]Bo;DENG [2,3]YuLin;QIN [2,3]WeiJie;QIAN [2,3]XiaoHong 机构地区：[1]School of Life Science and Technology,Beijing Institute of Technology;[2]National Center for Protein Sciences Beijing;[3]State Key Laboratory of Proteomics,Beijing Proteome Research Center,Beijing Institute of Radiation Medicine 出　　处：《Science China Chemistry》索引2014年第57卷第5期,共8页 基　　金：supported by the National Basic Research Program of China(2013CB911204);the National Key Scientific Instrument Development Program of China(2011YQ09000504);the National Natural Science Foundation of China(21275005,21235001) 摘　　要：Highly efficient and rapid proteolytic digestion of proteins into peptides is a crucial step in shotgun-based proteome-analysis strategy.Tandem digestion by two or more proteases is demonstrated to be helpful for increasing digestion efficiency and decreasing missed cleavages,which results in more peptides that are compatible with mass-spectrometry analysis.Compared to conventional solution digestion,immobilized protease digestion has the obvious advantages of short digestion time,no self-proteolysis,and reusability.We proposed a multiple-immobilized proteases-digestion strategy that combines the advantages of the two digestion strategies mentioned above.Graphene-oxide(GO)-based immobilized trypsin and endoproteinase Glu-C were prepared by covalently attaching them onto the GO surface.The prepared GO-trypsin and GO-Glu-C were successfully applied in standard protein digestion and multiple immobilized proteases digestion of total proteins of Thermoanaerobacter tengcongensis.Compared to 12-hour solution digestion using trypsin or Glu-C,14%and 7%improvement were obtained,respectively,in the sequence coverage of BSA by one-minute digestion using GO-trypsin and GO-Glu-C.Multiple immobilized-proteases digestion of the total proteins of Thermoanaerobacter tengcongensis showed 24.3%and 48.7%enhancement in the numbers of identified proteins than was obtained using GO-trypsin or GO-Glu-C alone.The ultra-fast and highly efficient digestion can be contributed to the high loading capacity of protease on GO,which leads to fewer missed cleavages and more complete digestion.As a result,improved protein identification and sequence coverage can be expected. 关 键 词：固定化蛋白酶 消化时间 超快速 氧化物 固定化胰蛋白酶 石墨 蛋白质组 试剂