硒的分析方法及泥鳅含硒蛋白的初步研究
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摘要
硒是人和动物体必需的微量元素之一,具有多种重要生理功能。我国一些地区早先发生的克山病和大骨节病就是由于当地居民长期摄硒不足而导致的。硒的有益性和致毒性之间的浓度界限很窄,建立灵敏度高、准确可靠的硒元素分析方法具有重要的现实意义。硒在动物体内主要通过与蛋白结合的形式来发挥它的功能性,分析鉴定含硒的蛋白可以使我们更好地了解硒对人体健康的影响机理。
本文建立了微波消解-氢化物原子荧光光谱法(MD-HG-AFS)和微波消解-电感耦合等离子体质谱法(MD-ICP-MS)两种硒含量分析方法,并对其进行了比较分析。两种方法在各自定量限上均能获得准确可靠的分析结果。MD-HG-AFS法样品处理相对繁琐,工作效率低,但在在试验成本上占据一定优势,利于普及推广。MD-ICP-MS法所需仪器昂贵,但样品制备简便快速,线性范围宽,检测灵敏度比前者高约20倍,分析速度快,适于大批量样品的多元素高通量分析。
在硒元素的形态分析上,建立了在线分离测定硒代胱氨酸(Se-Cys)和硒代蛋氨酸(Se-Met)的液相色谱-电感耦合等离子质谱法(LC-ICP-MS)和液相色谱-电喷雾串联质谱法(LC-ESI-MS-MS). LC-ICP-MS法中,Se-Cys和Se-Met在1-100μg/L均线性良好,两者的检出限分别为0.576μg/L和0.839μg/L,精密度试验中RSD值<10%,该方法能够满足快速检测Se-Cys和Se-Met含量的要求；LC-ESI-MS-MS法在优化条件下Se-Cys(定量子离子为248.1)和Se-Met(定量子离子为181.1)在2-100μg/L均线性良好,检出限分别为0.92μg/L和0.95μg/L。两种方法都能够应用于硒元素的形态分析,但前者在实际样品分析中基质干扰小,适于定量分析,而后者的定性分析则更加可靠,还可用于鉴定未知的含硒化合物,两种方法优势互补。
水产品中含有丰富的硒,采用已建立的分析方法对23种水产品中的硒含量进行了分析,最终以泥鳅为研究对象进一步分析。采用顺序提取法,依次对泥鳅肌肉中的水溶性蛋白、盐溶性蛋白、醇溶性蛋白和碱溶性蛋白进行了提取。结果未提到醇溶性蛋白,其它三者含量的相对百分比依次为碱溶性蛋白(63.27%)>水溶性蛋白(24.44%)>盐溶性蛋白(12.29%)。粗蛋白的分离纯化研究对比了两种阴离子交换填料(DEAE-Sephacel和DEAE sepharose FF),最终选用DEAE-Sephacel对粗蛋白提取物进行了初步分离纯化。经分离,从水溶性蛋白得到了W-1、W-2等六种蛋白组分；碱溶性蛋白分离得到了A-1、A-2等四个蛋白组分。采用SDS-PAGE分析,结果显示提取得到的蛋白样品分子量集中于4.1-66 kDa。由水溶性蛋白收集的六个组分中,W-5和W-6相对较纯,分子量分别处于35 kDa和20 kDa附近。W-1和W-2、W-3和W-4的分离效果欠佳,分别含有共同的蛋白组分。
硒在不同蛋白组分中分布情况的研究表明,三种蛋白粗提物中均含有一定的硒含量,含硒量依次为水溶性蛋白(1.866μg/g)>碱溶性蛋白(1.801μg/g)>盐溶性蛋白(1.607μg/g)；结合各蛋白粗提物的提取得率,可知蛋白硒占总硒的57.62%,可见硒在泥鳅肌肉中主要以与蛋白结合的形式存在,其中水溶性蛋白的W-4组分硒含量最高,为1.278μg/g。硒在泥鳅蛋白中的赋存形态研究中,对样品的水解条件进行了优化,证实酶解处理蛋白样品效果均好于盐酸水解,几种酶解方式中Ⅻ(胰蛋白酶+风味蛋白酶)的酶解效果最好。将LC-ICP-MS法和LC-ESI-MS-MS法结合分析证实,Se-Met是硒在所有蛋白样品中的唯一赋存形态。
采用胶内酶解处理、基质辅助激光解吸飞行时间串联质谱分析方式对硒含量相对较高的水溶性蛋白组分W-4进行了分析鉴定。检索结果表明,匹配度较高的三种蛋白均属于肌肉组织中肌酸激酶。
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.
本文建立了微波消解-氢化物原子荧光光谱法(MD-HG-AFS)和微波消解-电感耦合等离子体质谱法(MD-ICP-MS)两种硒含量分析方法,并对其进行了比较分析。两种方法在各自定量限上均能获得准确可靠的分析结果。MD-HG-AFS法样品处理相对繁琐,工作效率低,但在在试验成本上占据一定优势,利于普及推广。MD-ICP-MS法所需仪器昂贵,但样品制备简便快速,线性范围宽,检测灵敏度比前者高约20倍,分析速度快,适于大批量样品的多元素高通量分析。
在硒元素的形态分析上,建立了在线分离测定硒代胱氨酸(Se-Cys)和硒代蛋氨酸(Se-Met)的液相色谱-电感耦合等离子质谱法(LC-ICP-MS)和液相色谱-电喷雾串联质谱法(LC-ESI-MS-MS). LC-ICP-MS法中,Se-Cys和Se-Met在1-100μg/L均线性良好,两者的检出限分别为0.576μg/L和0.839μg/L,精密度试验中RSD值<10%,该方法能够满足快速检测Se-Cys和Se-Met含量的要求；LC-ESI-MS-MS法在优化条件下Se-Cys(定量子离子为248.1)和Se-Met(定量子离子为181.1)在2-100μg/L均线性良好,检出限分别为0.92μg/L和0.95μg/L。两种方法都能够应用于硒元素的形态分析,但前者在实际样品分析中基质干扰小,适于定量分析,而后者的定性分析则更加可靠,还可用于鉴定未知的含硒化合物,两种方法优势互补。
水产品中含有丰富的硒,采用已建立的分析方法对23种水产品中的硒含量进行了分析,最终以泥鳅为研究对象进一步分析。采用顺序提取法,依次对泥鳅肌肉中的水溶性蛋白、盐溶性蛋白、醇溶性蛋白和碱溶性蛋白进行了提取。结果未提到醇溶性蛋白,其它三者含量的相对百分比依次为碱溶性蛋白(63.27%)>水溶性蛋白(24.44%)>盐溶性蛋白(12.29%)。粗蛋白的分离纯化研究对比了两种阴离子交换填料(DEAE-Sephacel和DEAE sepharose FF),最终选用DEAE-Sephacel对粗蛋白提取物进行了初步分离纯化。经分离,从水溶性蛋白得到了W-1、W-2等六种蛋白组分；碱溶性蛋白分离得到了A-1、A-2等四个蛋白组分。采用SDS-PAGE分析,结果显示提取得到的蛋白样品分子量集中于4.1-66 kDa。由水溶性蛋白收集的六个组分中,W-5和W-6相对较纯,分子量分别处于35 kDa和20 kDa附近。W-1和W-2、W-3和W-4的分离效果欠佳,分别含有共同的蛋白组分。
硒在不同蛋白组分中分布情况的研究表明,三种蛋白粗提物中均含有一定的硒含量,含硒量依次为水溶性蛋白(1.866μg/g)>碱溶性蛋白(1.801μg/g)>盐溶性蛋白(1.607μg/g)；结合各蛋白粗提物的提取得率,可知蛋白硒占总硒的57.62%,可见硒在泥鳅肌肉中主要以与蛋白结合的形式存在,其中水溶性蛋白的W-4组分硒含量最高,为1.278μg/g。硒在泥鳅蛋白中的赋存形态研究中,对样品的水解条件进行了优化,证实酶解处理蛋白样品效果均好于盐酸水解,几种酶解方式中Ⅻ(胰蛋白酶+风味蛋白酶)的酶解效果最好。将LC-ICP-MS法和LC-ESI-MS-MS法结合分析证实,Se-Met是硒在所有蛋白样品中的唯一赋存形态。
采用胶内酶解处理、基质辅助激光解吸飞行时间串联质谱分析方式对硒含量相对较高的水溶性蛋白组分W-4进行了分析鉴定。检索结果表明,匹配度较高的三种蛋白均属于肌肉组织中肌酸激酶。
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.
引文
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