ABSTRACT
@#There are eight forms of vitamin E in human blood, including α-, β-, γ-, δ-tocopherols and α-, β-, γ-, δ-tocotrienols. As the most abundant and active form of vitamin E, α-tocopherol is widely accepted as a reliable indicator for nutritional assessment of body vitamin E status across the world. Considering that different vitamin E forms have diverse biological activities, separation and detection of different vitamin E forms in human blood facilitates the understanding of the association between vitamin E and diseases. In this review, the advances in sample-pretreatment techniques and detection techniques for vitamin E in human blood were presented. Currently, the sample-pretreatment techniques include solid-phase extraction, liquid-liquid extraction, dispersive liquid-phase microextraction, supported liquid extraction and direct protein precipitation; the detection techniques include automatic biochemical analysis, enzyme-linked immunosorbent assay, gas chromatography, liquid chromatography and ultra-high performance supercritical fluid chromatography mass spectrometry. This review summarizes the characteristics and scope of above-mentioned techniques used for detection of vitamin E in human blood, so as to provide insights into the selection of an appropriate method for inspection technicians.
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 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.