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| Optimization of enzymatic digestion conditions of shrimp allergens in mass spectrometry by response surface methodology |
| PAN Xiaodong, HUANG Baifen, CAI Zengxuan, WANG Mengli, HAN Jianlong, CHEN Qing
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| Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang 310051, China |
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Abstract Objective To optimize the enzymatic digestion conditions of trypsin, so as to improve the testing capacity of mass spectrometry for shrimp allergens. Methods The enzymatic digestion test was carried out by response surface methodology for optimizing pH, temperature and time. After enzymatic hydrolysis, the peptides were separated by chromatography and determined by high-resolution mass spectrometry with Q-orbitrap. The allergen protein was identified and quantified by UniProt database and MaxQuant software. Results Two allergen proteins, tropomyosin and arginine kinase, were isolated from shrimp, and their intensities ranged from 100.2×106 to 436.5×106. Response surface analysis showed that when the digestion time was 4.29 hours, the temperature was 44.15 ℃ and pH value was 6.55, the maximal intensity of the allergen proteins was 457.48×106. The experiment was validated with the digestion time of 4.2 h, pH value of 6.5, and temperature of 44 ℃, then resulted in the average intensity of 448.1×106. The deviation from the predicted value was 2.1%. Conclusion s The conditions of enzymatic digestion can be optimized by response surface methodology. The enzyme may have the best performance with the pH value of 6.5, temperature of 44 ℃ and digestion time of 4.2 hours.
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Received: 12 March 2021
Revised: 26 May 2021
Published: 10 August 2021
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