Bioassay

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

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  • 1. SCIEX China,Bejing 100015,China;
    2. Chongqing Institutes for Food and Drug Control,Chongqing 401121,China;
    3. 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

Received date: 2020-04-23

  Online published: 2024-05-31

Abstract

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.

Cite this article

ZHANG Ling-li, WANG Wen-tao, ZHAO Ying-hua, XIAO Zhi-liang, LUO Ji, LIU Bing, DONG Yan-dong, LI Xiang, CHEN Hong-xu . Analysis of glycopeptides in erythropoietin by CESI-MS/MS and NanoLC-MS/MS techniques*[J]. Chinese Journal of Pharmaceutical Analysis, 2021 , 41(1) : 79 -88 . DOI: 10.16155/j.0254-1793.2021.01.09

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