Studies On Analytical Methods For Selenium And Selenium-Containing Proteins In Misgurnus Anguillicaudatus

Selenium is an essential trace element for both human beings and animals, and it has a major nutritional and biological role in living systems. Evidences indicated that Keshan and Kashin-Beck diseases once occurred in some regions of China result from long-term insufficient intake of selenium in diet. There is a narrow range between the beneficial and toxic levels of selenium. So it is necessary to establish methods of high accuracy and precision in the measurement of selenium. Selenoproteins are the main biologically active form of selenium in organic systems, therefore identification and determination of these kinds of proteins is considered a better way to understand the influence of this element on human health.In this study, two different selenium analytical methods, MD-HG-AFS and MD-ICP-MS, were established and compared. The result showed that both of methods were accurate and reliable upon their each limit of quantitation (LOQ). The sample process method of MD-HG-AFS was complicated and ineffective. However, its low-cost advantage makes it easy to implement. Comparing with MD-HG-AFS, the MD-ICP-MS method has an advantage in analysis efficiency and multielement determination, but its high cost limits its application.In the analysis of selenium speciation two on-line separation and determination methods for selenoamino acids based on LC-ICP-MS and LC-ESI-MS-MS were established. With LC-ICP-MS, Se-Cys and Se-Met showed good linearity in the range of 1～100μg/L, the detection limit of Se-Cys and Se-Met were 0.576μg/L and 0.839μg/L, respectively, and RSD<10%. This method could satisfy the rapid detection of Se-Cys and Se-Met. In the optimized LC-ESI-MS-MS analysis, Se-Cys (quantitative ion is m/z 248.1) and Se-Met (quantitative ion is m/z 181.1) had good linearity in the range of 2～100μg/L, the detection limit for Se-Cys and Se-Met were 0.92μg/L and 0.95μg/L, respectively. Two methods can both be applied in the analysis of selenium speciation and complement each other. And the former one with little interference of matrix is suitable for quantitative analysis, while the later one is more dependable in qualitative analysis and also can be applied in identifying unknown compounds which contained selenium.Aquatic products are considered as an important source of selenium. In this paper, selenium content from 23 kinds of aquatic products was detected with established method, and loach was chosen as object for further analysis. Different extraction methods were used to extract the water-soluble protein, salt-soluble protein, alcohol-soluble protein, alkali-soluble protein in turn. The content of alkali-soluble protein, water-soluble protein and salt-soluble protein in loach muscle were 63.27%,24.44%,12.29%, respectively, but no alcohol-soluble protein was obtained from the sample. Then DEAE-Sephacel was chosen for the separation and purification of crude protein after two kinds of anion-exchange fillers were compared. After purification, W-1, W-2, W-3, W-4, W-5, W-6 were collected from water-soluble protein, and A-1, A-2, A-3, A-4 from alkali-soluble protein. After SDS-PAGE analysis, the result showed that MW of protein in samples were mainly between 4.1～66 kDa. W-5 and W-6 were almost single-banded and the molecular weights were about 35 kDa,20 kDa respectively, but W-1, W-2, W-3, W-4 were protein mixtures.The selenium distribution among different protein fractions was studied. It showed that all of the three crude protein extracts contained selenium, and the contents of selenium were water-soluble protein (1.866μg/g)>alkali-soluble protein (1.801μg/g)>salt-soluble protein (1.607μg/g). Taking the extraction efficiency into account,57.62%of total selenium was in the form of selenoproteins or selenium-containing proteins. W-4 contained 1.278μg se/g with the highest selenium content. By comparing two different sample preparation strategies, the enzymatic digestion was much more effective. Among four different enzymatic hydrolysis (Ⅸ-Ⅻ) processes, highest degradation efficiency was achieved with the combination of trypsin and flavourzyme (Ⅻ). Both of the methods, LC-ICP-MS and LC-ESI-MS-MS, were applied to detect selenium speciation. Se-Met was the only selenoamino acid form in all samples.MALDI TOF/TOF analysis followed in-gel digestion was applied to identifying W-4, which had relatively higher selenium content. A database search was carried out in the non-redundant NCBI database. The three identified proteins were all creatine kinases derived from muscle tissues.