超声辅助胶内酶切方法的优化及其酶切特点的分析  

Optimization of Ultrasound-Assisted In-Gel Digestion Method and Analysis of Peptide Characteristics

作　　者：肖晓萍[1] 郭正光[1] 成洁[1] 孙伟[1] 

机构地区：[1]中国医学科学院基础医学研究所北京协和医学院基础学院中心实验室,北京100005

出　　处：《质谱学报》2017年第2期187-194,共8页Journal of Chinese Mass Spectrometry Society

基　　金：国家重点基础研究发展计划(2013CB530805、2014CBA02005);中国科技部重点基础研究发展计划(2013FY114100)资助

摘　　要：分别应用20、100和200 W超声功率对牛血清白蛋白以及人尿蛋白组的胶内条带进行胶内酶切,通过比较不同功率鉴定的多肽数和蛋白数,确定最优的超声功率。同时比较超声辅助酶切与传统过夜酶切方法,并分析超声辅助胶内酶切方法的特点。结果表明,超声功率为20W时对胶粒破坏最小,样品损失最少,在牛血清白蛋白样品中鉴定的多肽数最多为65。在人尿蛋白组2个条带中,20 W方法鉴定的蛋白数分别为168和199,鉴定效果最优。通过对多肽误切率和不完全酶切多肽的定性和定量分析发现,超声酶切方法的误切率高于传统方法,但不完全酶切率较低。通过分析多肽误切位点发现,传统过夜酶切法和超声辅助酶切法的主要误切类型分别为脯氨酸(P)和天冬氨酸/谷氨酸(D/E),3种超声方法之间各种误切类型所占比例均无明显差别。超声辅助酶切可显著缩短蛋白质胶内酶切时间,其中20 W功率超声酶切的效果最佳。与传统过夜酶切法相比,超声辅助酶切多肽的鉴定结果更好,其多肽的误切率更高,不完全酶切率较低,可用于发现蛋白质组学研究。Bovine serum albumin and human urinary protein gel bands of two different molecular weight were digested by 20 W, 100 W and 200 W ultrasound-assisted in-gel digestion method, and the digested peptides were analyzed by high-resolution mass spectrometry. By comparing the number of identified proteins and peptides, the optimal ultrasonic power for ultrasound-assisted in-gel digestion was determined. Meanwhile, the peptide characteristics of ultrasound-assisted in-gel digestion method were analyzed by comparing with traditional overnight digestion method. 20 W ultrasound-assisted in-gel digestion method shows the minimal gel damage and loss for in-gel protein samples, the number of peptides identified in BSA is 65, while the number of identified peptides from 100 W, 200 W and the traditional overnight digestion methods is 57, 51 and 41, respectively, much lower than 20 W ultrasound-assisted in-gel digestion method. The number of proteins identified in two different gel bands of human urinary protein in 20 W ultrasound-assisted in-gel digestion method is 168 and 199, respectively, which is better than 100 W, 200 W and the traditional overnight digestion method. By analyzing the mis-cleavage rate and semi-tryptic rate of peptides based on the qualitative and quantitative analysis, the mis-cleavage rate of ultrasound-assistedin-gel digestion is higher than traditional overnight digestion method, while the semi-tryptic rate of ultrasound-assisted in-gel digestion is lower. The mis-cleavage type analysis of identified peptides indicates that traditional overnight method can generate more Proline （P） type mis-cleavage peptides, and ultrasound-assisted in-gel digestion method produces more aspartate/glutamate （D/E） type mis-cleavage peptides. The mis-cleavage peptide types from three ultrasound powers show no significant differences. Compared with traditional overnight digestion method, ultrasound-assisted in-gel digestion method can significantly shorten in-gel protein digestion-time and 20 W ultrasonic power shows

关 键 词：蛋白质组 胶内酶切 超声辅助酶切 

分 类 号：O657.63[理学—分析化学]