蛋白质多肽酶解及分离新技术的改进  

Improvement of enzymolysis and separation technology in protein

作　　者：刘宝阳 张磊[2] 范志娟[2] 田亚琼[2] 刘树业[2] LIU Baoyang;ZHANG Lei;FAN Zhijuan;TIAN Yaqiong;LIU Shuye(Department of Clinical Laboratory,Tianjin Baodi Hospital/Baodi Clinical Collegeof Tianjin Medical University,Tianjin 301800,China;Department of Clinical Laboratory,Third Central Hospital of Tianjin,Tianjin 300170,China)

机构地区：[1]天津市宝坻区人民医院检验科/天津医科大学宝坻临床学院,天津301800 [2]天津市第三中心医院检验科,300170

出　　处：《检验医学与临床》2018年第23期3570-3572,共3页Laboratory Medicine and Clinic

摘　　要：目的探讨新型蛋白质多肽酶解和分离技术,以此提高蛋白质的质谱鉴定水平,使其适用于更复杂生物样品,从而弥补传统蛋白质组学利用的二维电泳-质谱技术所产生的不足。方法利用美国promega和自主修饰Self-owned胰蛋白酶,酶解浓度不同的牛血清清蛋白测定总离子流图(TIC)并通过Proteome Discoverer软件进行数据分析,测定牛血清清蛋白覆盖率。利用Self-owned胰蛋白酶酶解牛血清清蛋白,其中酶切液用自制的二维一体毛细管色谱柱进行分离,通过高效液相色谱与质谱技术平台,通过对TIC、信号强度和保留时间(RT),观察毛细管色谱柱分离性能和重复性。结果美国promega和Self-owned胰蛋白酶酶解浓度不同的牛血清清蛋白结果显示,Self-owned胰蛋白酶在高效液相色谱与质谱技术鉴定高、中、低浓度牛血清清蛋白覆盖率分别为38.22%、28.34%、13.18%;而美国promega胰蛋白酶酶解牛血清清蛋白覆盖率分别为34.93%、26.36%、10.21%。Self-owned胰蛋白酶和自制的二维一体毛细管色谱柱联用,用于牛血清清蛋白分析,通过质谱分析,结果显示,在极低浓度条件下,牛血清清蛋白的覆盖率是24.22%。在相同的实验条件下重复进行两次实验,观察自制的二维一体毛细管色谱柱的重复性,结果显示选用牛血清清蛋白酶切的肽段混合物中质荷比(m/z)740.4为强度较高的特征肽段,两次的RT是51.90、51.95 min,信号强度是6.77×10~5、7.42×10~5。结论 Self-owned胰蛋白酶拥有高催化活性,自制的二维一体毛细管色谱柱具有较高的分离性能和重复性,将两者联合应用于蛋白质组学技术中,可以显著增高蛋白质数量的检出率。Objective To discuss enzymolysis and separation technology in protein,so as to improve the level of proteins identified by mass spectrometry. Methods With Promega and Self-owned trypsin digestion of different concentrations of bull serum albumin （BSA）,total ion chromatogram （TIC） were measured. And by Proteome Discoverer software,data processing was analyzed to measure BSA coverage rate. Digested with Self-owned trypsin BSA,two-dimensional liquid enzyme used in this study prepared in one of the capillary column was separated by high performance liquid chromatography and mass spectrometry （nanoHPLC-MS） technology platform. And its capillary column separation performance and reproducibility were measured through TIC,retention time （RT） and signal strength. Results With promega and Self-owned trypsin digestion of different concentrations of BSA,high,medium and low concentrations of BSA coverage rates identified by nanoHPLC-MS technique were 38.22%,28.34% and 13.18%,while,which identified by Promega tryptic were 34.93 %,26.36% and 10.21%. Under the condition of the Self-owned trypsin combined with one-dimensional capillary column for the identification of BSA,and by LTQ-Orbitrap XL mass spectrometer,identification BSA coverage rate was 24.22%,which was at very low concentrations. Under the same experimental conditions,one-dimensional capillary column reproducibility was observed. Mixture of mass-to-charge ratio（m/z）of 740.4 peptides,twice RT of BSA peptides were 51.90 min and 51.95 min,and signal strength were 6.77×10 5 and 7.42× 10 5 respectively. Conclusion Self-owned trypsin shows a high catalytic activity,and one-dimensional capillary column has a better separation performance and reproducibility.Combined detection of these two methods in the proteome could improve the detection rate of amount of proteins.

关 键 词：蛋白质组学 胰蛋白酶 二维一体毛细管色谱柱 

分 类 号：R446[医药卫生—诊断学]