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1 650 V,MS 扫描范围为m/z 350~1 250,MS/MS 扫描范围为m/z 100~1 500。NanoLC-MS/MS 分析方法：采用YMC C18 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5 μL·min-1;离子源温度300 ℃,喷雾电压为+5 500 V,MS 扫描范围为m/z 350~1 250,MS/MS 扫描范围为m/z 100~1 500。将采集到的质谱数据导入到软件BiopharmaViewTM3.0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。
张伶俐, 王文涛, 赵颖华, 肖志良, 罗继, 刘冰, 董衍东, 李响, 陈泓序
. CESI-MS/MS和NanoLC-MS/MS技术对促红细胞生成素中糖肽的分析*[J]. 药物分析杂志, 2021
, 41(1)
: 79
-88
.
DOI: 10.16155/j.0254-1793.2021.01.09
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 1 650 V,MS scanning range m/z 350-1 250,MS/MS scanning range m/z 100-1 500 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,1.7 μm） 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 5 μL·min-1. The mass spectrometer was in positive ion and IDA modes to collect data. The ion source temperature 300 ℃,the spray voltage +5 500 V, MS scanning range m/z 350-1 250,and MS/MS scanning range m/z 100-1 500 were used in LC-MS/MS analysis. The acquired data were imported into the software BiopharmaViewTM 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 the characterization of glycoproteins,CESIMS/ MS and NanoLC-MS/MS provide complementary results for the analysis of glycopeptides. The combination of these methods can obtain more glycopeptides information and make more accurate quantification,providing a more comprehensive solution for glycoproteins analysis.
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