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.
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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