Shangguan Lulu, Zhang Lingyi, Xiong Zhichao, Ren Jun, Zhang Runsheng, Gao Fangyuan, Zhang Weibing
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, P.R. China.
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, P.R. China.
J Chromatogr A. 2015 Apr 3;1388:158-66. doi: 10.1016/j.chroma.2015.02.040. Epub 2015 Feb 17.
The bottom-up strategy of proteomic profiling study based on mass spectrometer (MS) has drawn high attention. However, conventional solution-based digestion could not satisfy the demands of highly efficient and complete high throughput proteolysis of complex samples. We proposed a novel bi-enzymatic reactor by immobilizing two different enzymes (trypsin/chymotrypsin) onto a mixed support of hybrid organic-inorganic monolith with SBA-15 nanoparticles embedded. Typsin and chymotrypsin were crossly immobilized onto the mixed support by covalent bonding onto the monolith with glutaraldehyde as bridge reagent and chelation via copper ion onto the nanoparticles, respectively. Compared with single enzymatic reactors, the bi-enzymatic reactor improved the overall functional analysis of membrane proteins of rat liver by doubling the number of identified peptides (from 1184/1010 with trypsin/chymotrypsin enzymatic reactors to 2891 with bi-enzymatic reactor), which led to more proteins identified with deep coverage (from 452/336 to 620); the efficiency of the bi-enzymatic reactor is also better than that of solution-based tandem digestion, greatly shorting the digestion time from 24h to 50s. Moreover, more transmembrane proteins were identified by bi-enzymatic reactor (106) compared with solution-based tandem digestion (95) with the same two enzymes and enzymatic reactors with single enzyme immobilized (75 with trypsin and 66 with chymotrypsin). The proteolytic characteristics of the bi-enzymatic reactors were evaluated by applying them to digestion of rat liver proteins. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weight.
基于质谱仪（MS）的蛋白质组分析研究的自下而上策略备受关注。然而，传统的基于溶液的酶解方法无法满足复杂样品高效、完全高通量蛋白水解的需求。我们提出了一种新型双酶反应器，将两种不同的酶（胰蛋白酶/糜蛋白酶）固定在嵌入SBA - 15纳米颗粒的有机 - 无机混合整体支撑体上。胰蛋白酶和糜蛋白酶分别通过戊二醛作为桥联试剂共价结合到整体支撑体上以及通过铜离子螯合到纳米颗粒上的方式交叉固定在混合支撑体上。与单酶反应器相比，双酶反应器将鉴定出的肽段数量翻倍（从胰蛋白酶/糜蛋白酶酶解反应器的1184/1010增加到双酶反应器的2891），从而提高了大鼠肝脏膜蛋白的整体功能分析，这使得更多具有深度覆盖的蛋白质被鉴定出来（从452/336增加到620）；双酶反应器的效率也优于基于溶液的串联酶解，将酶解时间从24小时大幅缩短至50秒。此外，与使用相同两种酶的基于溶液的串联酶解（95种）和固定单一酶的酶解反应器（胰蛋白酶75种，糜蛋白酶66种）相比，双酶反应器鉴定出了更多的跨膜蛋白（106种）。通过将双酶反应器应用于大鼠肝脏蛋白质的酶解来评估其蛋白水解特性。该反应器对不同疏水性和分子量的蛋白质均显示出良好的酶解能力。
