Optimization of enzymatic digestion conditions of shrimp allergens in mass spectrometry by response surface methodology / 预防医学

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.

Analysis of allergen protein in Macrobrachium by ultra-high performanceliquid chromatography coupled with quadrupole orbitrap mass spectrometry / 预防医学

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.

Structures and Antigenic Epitopes of Dust Mite Allergens: Der p 2 and Der f 2: a Comparative Study / 现代医院

Objective To compare and analyze the primary and secondary structures and antigenic epitopes of the two allergens: Der p 2 and Der f 2. Methods The protein sequences of Der p 2 and Der f 2 were downloaded online. The primary and secondary structures of the dust mite allergens were compared and analyzed bioinformatically to determine the potential epitope and signal peptide sites. Results Both Der p 2 and Der f 2 contained 146 amino acids and 9 potential protein binding sites with a secondary structure that mainly contains [3 - sheets, and there might be signal peptides site at the 1st 17th segment of the N - terminus. B cell epitopes analysis revealed that both Der p 2 and Der f 2 have 9 potential linear B epitopes and 2 conformational B epitopes. NetMHCⅡserver prediction showed Der p 2 contains 6 high affinity sites, whereas Der f 2 0nly contains 5. Conclusion This study may lay the foundation for further research of the biochemical function of the 2 allergens and contribute to vaccine development for allergen - specific immunotherapy.

Identification and purification of major allergens in Artemisia sieversiana pollen / 中国免疫学杂志

Objective:To isolate,identify and purify the Artemisia sieversiana pollen ,the mostly widespread pollen among the Artemisia pollens in China.Methods: Artemisia sieversiana extract was precipitated by saturated ammonium sulfate and then electrophoresed by SDS-PAGE.The molecular mass of each protein band was determined by gel media system.The primary allergen proteins were identified by Western blot.Allergen proteins were purified and identified by DEAE-cellulose DE-52 ion exchange chroma-tography ( IEC) and Western blot.Results: We isolated more than twenty protein bands from Artemisia sieversiana pollen extract , including the most abundant six bands whose Mr were 62 kD,57 kD,38 kD,29 kD,25 kD,14 kD espectively.The protein bands with Mr were 62 kD and 16 kD had the highest binding capacity with the specific IgE from Artemisia pollen allergic patients.The DEAE-cellulose DE-32 IEC was used to purify the primary allergen proteins with Mr 62 kD and 16 kD.Conclusion:The primary allergens of Artemisia sieversiana include the allergen proteins whose Mr are 62 kD,16 kD and the allergen of Mr 62 kD and 16 kD can be purified by chromatography.