【摘要】 目的:制备高纯度、酶解效率高、酶切位点专一的测序级胰蛋白酶,应用于蛋白组学研究的蛋白质鉴定与分析中。方法:取实验室自制的猪胰蛋白酶粗酶,经硼氢化钠、甲醛还原甲基化修饰抑制胰蛋白酶自水解,采用高效液相色谱仪15RPC反相柱纯化,收集对应的甲基化胰蛋白酶峰组分,冷冻干燥;甲基化修饰的胰蛋白酶进一步经甲苯磺酰苯丙氨酰氯甲酮(TPCK)修饰,以抑制糜蛋白酶等非特异性酶切活性,并经反相色谱柱再纯化,获得终产物即质谱测序级胰蛋白酶;自制的测序级胰蛋白酶经SDS-PAGE、HPLC反相色谱分析、酶比活力测定,并应用于胶内蛋白质酶切质谱鉴定氨基酸序列等,检测其纯度、酶水解效率及酶切位点特异性。结果:自制甲基化TPCK修饰的测序级胰蛋白酶纯度大于95%,酶比活力为200 U/mgP(TAME)以上,质谱分析酶切特异性好;且酶的制备工艺流程稳定,可应用于测序级胰蛋白酶产品的生产与开发中。结论:制备的测序级胰蛋白酶纯度高、酶解效率优、酶切特异性强,可广泛应用于实验室中蛋白质和肽段测序鉴定、HPLC肽段谱图分析等蛋白组学研究分析中。更多还原

【Abstract】 Objective: To prepare highly purified, effective, restriction site-specific and chemically stabilized sequencing grade modified trypsin, and apply for protein identification and analysis in proteomics research. Methods: Firstly, raw trypsin isolated from porcine pancreas was reductive methylated with sodium borohydride buffer and formaldehyde following purification by reverse phase chromatography of 15RPC column, producing a stable trypsin that was resistant to tryptic autolysis. Then, the chromatography peak composition of methyl-trypsin was collected. Secondly, methyl-trypsin was further improved by TPCK treatment and was subjected to extensive purification by reverse phase chromatography to remove other non-specific enzyme activity from contaminating proteases. The final product was sequencing grade modified trypsin. Thirdly, the quality of sequencing grade modified trypsin was tested by SDS-PAGE, HPLC reversed phase chromatography, catalytic activity on TAME, typical enzyme substrate and characterized by assay which related to its application in-gel tryptic digestions following identified by mass spectrometry. Results: Purity of sequencing grade modified trypsin was more than 95% in HPLC assay, appeared as a single band on SDS-PAGE. The specific activity was 200 TAME U/mgP above. The highly purified and chemically stabilized sequencing grade trypsin gave excellent performance for using in-gel tryptic digestions. And the preparation process of enzyme was stably and repeatable. Conclusion: This process yielded a highly purified trypsin product that was effective, highly specific cleavage resulting in a limited number of tryptic peptides and stable, can be widely used in proteomics for peptide mapping and protein identification work at lab. And the stable preparation process can be applied to produce and develop mass spectrum sequencing grade modified trypsin.更多还原