Citation: WANG Jian-Wei, ZHAO Li, TIAN Pu. Sequence Analysis of Insulin Degludec by Top-Down High Resolution Tandem Mass Spectrometry[J]. Chinese Journal of Analytical Chemistry, ;2020, 48(8): 1104-1110. doi: 10.19756/j.issn.0253-3820.201138 shu

Sequence Analysis of Insulin Degludec by Top-Down High Resolution Tandem Mass Spectrometry

WANG Jian-Wei ^1,2, ,
ZHAO Li ¹ ,
TIAN Pu ¹

1.
School of Life Sciences, Jilin University, Changchun 130012, China;
2.
National Analytical Research Center of Electrochemistry and Spectroscopy, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China

Received Date: 17 March 2020
Revised Date: 17 April 2020

The "top-down" high-resolution tandem mass spectrometry method was used for the first time to comprehensively analyze the amino acid sequence of insulin degludec and the non-protein modification sequence of C-terminal side chain of B-chain. The reduction conditions of insulin degludec and the collision conditions of tandem mass spectrometry were optimized. The results indicated that insulin degludec could be fully reduced to two independent peptide chains when tris(2-carboxyethyl) phosphine (50 mmol/L) and guanidine hydrochloride (6 mol/L) were used in combination and reacted for 40 min at 45℃. The reduction products were separated by Accucore C₁₈ column and analyzed by orbitrap high resolution tandem mass spectrometry. When the higher energy collision induced dissociation was 20 for Chain A and 25 for Chain B, the most abundant fragment information could be obtained, which not only met the basic requirements of 100% peptide coverage in biotechnology drug evaluation, but also provided more comprehensive sequence analysis for the problem samples. This method eliminated the costly enzymatic hydrolysis step in the traditional sequence analysis, limited the risk of introducing artifactual modification to obtain better sequence information, thus saved the cost and significantly improved work efficiency, and provided a new solution for sequence analysis of insulin analogs such as insulin degludec.
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