CESI-MS/MS和NanoLC-MS/MS技术对促红细胞生成素中糖肽的分析  

Analysis of glycopeptides in erythropoietin by CESI-MS/MS and NanoLC-MS/MS techniques

作　　者：张伶俐 王文涛 赵颖华 肖志良 罗继 刘冰 董衍东 李响[3] 陈泓序 ZHANG Ling-li;WANG Wen-tao;ZHAO Ying-hua;XIAO Zhi-liang;LUO Ji;LIU Bing;DONG Yan-dong;LI Xiang;CHEN Hong-xu(Chongqing Institutes for Food and Drug Control,Chongqing 401121,China;SCIEX China,Bejing 100015;National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Beijing 100050,China)

机构地区：[1]重庆市食品药品检验检测研究院,重庆401121 [2]SCIEX中国,北京100015 [3]中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京100050

出　　处：《药物分析杂志》2021年第1期79-88,共10页Chinese Journal of Pharmaceutical Analysis

基　　金：国家药典委员会药品标准制修订研究课题:重组人促红素标准提高(课题编号2019S008)。

摘　　要：目的:使用无鞘液毛细管电泳-质谱联用(CESI-MS/MS)和纳升液相色谱-质谱联用(NanoLCMS/MS)2种方法,对高糖基化蛋白促红细胞生成素(EPO)的糖肽进行分析,并对2种方法的分析结果进行对比。方法:以EPO为研究对象,还原烷基化后用胰蛋白酶进行酶切,酶切得到的肽段利用CESIMS/MS和NanoLC-MS/MS检测方法进行分离鉴定。CESI-MS/MS分析方法:采用熔融的石英毛细管,以10%醋酸水溶液为背景电解质,在34.5 kPa压力下进样60 s,分离电压为30 kV,同时施加正向压力6.9kPa;离子源温度为50℃,喷雾电压为1650 V,MS扫描范围为m/z 350~1250,MS/MS扫描范围为m/z 100~1500。NanoLC-MS/MS分析方法:采用YMC C_(18)色谱柱,以含0.1%甲酸的水溶液(A)-含0.1%甲酸的乙腈溶液(B)为流动相,梯度洗脱,流速5μL·min^(-1);离子源温度300℃,喷雾电压为+5500 V,MS扫描范围为m/z 350~1250,MS/MS扫描范围为m/z 100~1500。将采集到的质谱数据导入到软件BiopharmaView^(TM)3.0中进行搜库检索,对糖肽进行鉴定。结果:采用CESI-MS/MS方法可以单独鉴定到32条糖肽,采用NanoLC-MS/MS方法可以单独鉴定到40条糖肽;通过2种方法可以共鉴定到84条糖肽,共涉及56种糖型。对比2种方法的结果,发现对于EPO糖肽的分析,CESI-MS/MS方法可以鉴定到更多的含唾液酸的糖肽,并表现了更优异的分离效果。此外,CESI-MS/MS方法鉴定出的糖肽分子量范围更宽,可以有效分析NanoLC-MS/MS方法中未鉴定到的高分子量糖肽。结论:在糖蛋白的分析中,通过糖肽水平的分离和鉴定,可以获得糖基位点和糖基序列的信息。CESI-MS/MS方法和NanoLC-MS/MS方法提供了互补的分析结果,其联合使用可鉴定出更多种类的糖肽,为糖蛋白的研究中糖肽水平的分析提供了更全面的解决方案。Objective:To analyzed and compare the glycopeptides of high glycosylated protein erythropoietin(EPO)by sheathless capillary electrophoresis-mass spectrometry(CESI-MS/MS)and nano-liquid chromatography mass spectrometry(NanoLC-MS/MS).Methods:EPO was used as the object of study.After reduction and alkylation,EPO was digested by trypsin.The digested peptides were separated and identified by CESI-MS/MS and NanoLC-MS/MS method.CESI-MS/MS analysis:Capillary electrophoresis was performed in fused silica capillary with 10%acetic acid aqueous solution as background electrolyte.Samples were injected for 60 s at 34.5 kPa pressure.The separation voltage was 30 kV with 6.9 kPa forward pressure.The ion source temperature 50℃,spray voltage 1650 V,MS scanning range m/z 350-1250,MS/MS scanning range m/z 100-1500 were used in CESI-MS/MS analysis.LC-MS/MS analysis:The chromatographic separation was carried out on a C18 column(2.1 mm×150 mm,1.7μm)with the mobile phase consisting of water containing 0.1%formic acid(A)-acetonitrile containing 0.1%formic acid(B)in a gradient mode at the flow rate of 5μL·min^(-1).The mass spectrometer was in positive ion and IDA modes to collect data.The ion source temperature 300℃,the spray voltage+5500 V,MS scanning range m/z 350-1250,and MS/MS scanning range m/z 100-1500 were used in LC-MS/MS analysis.The acquired data were imported into the software BiopharmaView^(TM) 3.0 to get the glycopeptides information.Results:32 glycopeptides were solely identified by CESI-MS/MS,while 40 glycopeptides were solely identified by NanoLC-MS/MS.A total of 84 glycopeptides and 56 glycans were identified by both methods.Comparing the results in different methods,it was found that CESI-MS/MS method could identify more sialic acid-containing glycopeptides and showed better separation efficiency for glycopeptides in EPO.In addition,CESI-MS/MS identified glycopeptides which had a wider range of molecular weight and could effectively analyze the glycopeptides which were not identified in NanoLC-MS/MS.Conclusion:In

关 键 词：促红细胞生成素 糖肽 糖蛋白激素 糖基化修饰 无鞘液毛细管电泳-质谱联用 纳升液相色谱-质谱联用 

分 类 号：R917[医药卫生—药物分析学]