Citation: LIU Hai-Long, REN Wei-Cheng, ZONG Li, WANG Ying-Wu. Pepsin Digestion Combined With Post-Column Online Reduction for Analysis of Disulfide Bonds in Recombinant Human Granulocyte Stimulating Factor[J]. Chinese Journal of Analytical Chemistry, ;2019, 47(4): 508-518. doi: 10.19756/j.issn.0253-3820.181806 shu

Pepsin Digestion Combined With Post-Column Online Reduction for Analysis of Disulfide Bonds in Recombinant Human Granulocyte Stimulating Factor

School of Life Sciences, Jilin University, Changchun 130012, China

Received Date: 26 December 2018
Revised Date: 21 February 2019

A disulfide bond analysis method was presented in this study, which digested protein with pepsin, and combined post-column reduction with liquid chromatography-tandem mass spectrometry for peptide analysis. When protein was digested in an acidic environment, the original conformation of the disulfide bond could be maintained as much as possible; the method of post-column online reduction could make up for the poor specificity of pepsin and difficulty in data analysis. This method was used to analyze the disulfide bonds of recombinant human granulocyte-colony stimulating factor (rhG-CSF), and the results showed that disulfide bonds in NEM treated rhG-CSF were Cys36-Cys42 and Cys64-Cys74, leaving Cys17 alkylated by NEM, and no scrambling signal was detected. When analyzing the disulfide bond of rhG-CSF by this method without alkylating, the scrambling ratios were 9.1% (Cys36-Cys42), 0%(Cys64-Cys74) and 12.6% (Cys17), the results obtained from traditional method with chymotrypsin and Glu-C + trypsin were 73.4%, 58.1%, 97.5% and 40.3%, 5.2%, 22.3% correspondingly. Compared with the conventional method, this method showed a lower disulfide bond scrambling ratio, the measurement could more accurately represent the actual existence state of the disulfide bond in the protein.
- Pepsin,
- Disulfide bonds,
- Post-column online reduction,
- Liquid chromatography-tandem mass spectrometry,
- Recombinant human granulocyte stimulating factor
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