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%.@*Conclusions@#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.
ABSTRACT
Objective @#To develop an analytical method of ibotenic acid (IBA) and muscimol (MUS) in wild mushroom by dansyl chloride (DNSCl) derivatization-liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to provide technical support for etiological identification of mushroom poisoning events.@*Methods @#The sample was extracted with hydrochloric acid solution, derived by bimolecular DNSCl, diluted and inorganic salts precipitated with acetonitrile. The extract was separated by a waters XBridgeTM BEH C18 column and measured by LC-MS/MS.@*Results @#The limits of detection for IBA and MUS in wild mushroom were 0.15 mg/kg and 0.1 mg/kg, respectively. Good linear relationship was obtained for IBA and MUS at the range of 0.5-250 mg/kg with the correlation coefficient of 0.997 and 0.999, respectively. The average recoveries at three spiking levels were 84.5%-102.0% with relative standard deviations (RSDs, n=6) of 4.7%-8.6% for IBA. The average recoveries were 88.6%-95.4% with RSDs (n=6) of 4.9%-7.5% for MUS. @*Conclusion @#The optimized sample extraction and bimolecular DNSCl derivatization conditions can achieve rapid and accurate analysis of IBA and MUS in wild mushroom poisoning sample.
ABSTRACT
Objective@#To establish the ultra-high performance liquid chromatography coupled with quadrupole orbitrap mass spectrometry ( UPLC-Q-Orbitrap-MS ) for the analysis of allergen protein in Macrobrachium. @*Methods@#Based on the strategy of bottom-up protein analysis, the proteins in Macrobrachium samples were extracted by Tris-HCl, digested by trypsin at 40 ℃ for 6 hours, separated by chromatography, and analyzed by Q-Orbitrap-MS spectrometry ( Full MS/dd-MS2, TopN=10 ) . Allergen proteins were identified with UniProt protein database and Proteome Discoverer software. @*Results@# Four kinds of allergen proteins were obtained, which were tropomyosin, arginine kinase, sarcoplasmic calcium binding protein and hemocyanin. The coverage rates of peptides in proteins were 53%, 36%, 12% and 12%, respectively. Post translation modifications were methylation of aspartic acid (D), deacylylation of aspartic acid ( N ) , glutamyl ammonia ( Q ) and oxidation of methionine ( M ) .@*Conclusions@#The UPLC-Q-Orbitrap-MS can identified abundant peptide and fragment information with high sensitivity and resolution, which provides a technical support for the analysis of shrimp allergens.