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.

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.

The purpose of this study was to investigate the damage mechanism of pathogenic E.coli on mouse brain microvascular endothelial cells(BMEC cells)and mouse alveolar macrophages(MH-S cells),as well as the lung and brain of healthy mice.In this study,BMEC cells and MH-S cells were infected with pathogenic E.coli strains,and cell morphological changes were observed.Plate counting method was used to detect the adhesion and invasion ability of the strains to cells and the number of bacteria in the lungs and brains of mice.RT-qPCR was used to detect the ex-pression of TNF-α,IL-1β and IL-6 genes in cells and mouse organs at different time periods.West-ern blot was used to detect the expression of p-NF-κB,p-JAK2 and p-STAT3 proteins related to inflammation in cells and mouse organs after infection.The results showed that the cell culture medium of the infection group was turbid,the cell vision became dark and blurred,some cells shrank and died,and more fragments were produced.The adhesion rate and invasion rate of BMEC cells at 3 h were significantly lower than those at 6 h(P＜0.050),and the adhesion rate and inva-sion rate of MH-S cells at 3 h were significantly higher than those at 6 h(P＜0.010).Infected mice had a large area of swelling and bleeding in the brain,and the lungs had different degrees of swell-ing and bleeding.The bacterial load in the brain and lung was the highest at 12 h.Compared with the control group,the mRNA expression levels of IL-1β,IL-6 and TNF-α in the infection group were significantly increased at 3 h and 6 h(P＜0.050),and the mRNA expression levels of inflam-matory factors in BMEC cells and MH-S cells were the highest at 6 and 3 h,respectively.The mR-NA expression of inflammatory factors in the brain and lung of infected mice showed a trend of in-creasing first and then decreasing with time,with the highest expression at 12 h after infection.The expression levels of p-NF-κB protein in BMEC cells,MH-S cells,lung and brain tissues of mice in the infection group were significantly higher than those in the control group(P＜0.001),and the expression levels of p-JAK2 protein and p-STAT3 protein were significantly lower than those in the control group(P＜0.050).The above results showed that pathogenic E.coli could adhere and invade BMEC cells and MH-S cells,colonize in lung and brain tissues of mice,promote the expres-sion of NF-κB protein in cells and tissues,inhibit the expression of JAK2 protein and STAT3 pro-tein,and then stimulate cells and tissues to produce inflammatory response.