Modification of EDC method for increased labeling efficiency and characterization of low-content protein in gum acacia using asymmetrical flow field-flow fractionation coupled with multiple detectors.
Modification of EDC method for increased labeling efficiency and characterization of low-content protein in gum acacia using asymmetrical flow field-flow fractionation coupled with multiple detectors.
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DOI：
10.1007/s00216-021-03587-y
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发表时间：
2021-10
影响因子：
4.3
通讯作者：
Choi J
中科院分区：
文献类型：
--
作者：
Zhang M;Nilsson L;Lee S;Choi J
1-乙基-3-(3-二甲氨基丙基)碳二亚胺(EDC)在生物化学和食品分析领域被广泛用作蛋白质荧光标记的交联剂。许多天然多糖往往含有一些蛋白质或多肽，含量很低，但在其生物学功能和技术应用中发挥着至关重要的作用。对这些低含量蛋白质物质的测定需要一种高度灵敏和选择性的方法。在这项研究中，建立了一种方法学的方法来研究蛋白质物质的存在对多糖的摩尔质量分布(MD)以树胶(GA)为模型多糖。对EDC荧光标记法进行了改进，改变溶液的pH值(7、9、11)，用于食品原料中低含量蛋白质的分析。用荧光光谱分析和不对称流动分馏(AF4)表征了未标记和荧光标记GA的标记效率和物理化学性质。AF4提供了阿拉伯半乳糖(AG)和阿拉伯半乳糖蛋白复合体(AGP)的摩尔质量(M)和旋转半径(Rg)，并确定了MD上蛋白质类物质的存在。荧光光谱分析表明，在pH为7、9和11时，GA的标记率分别为56.5%、68.4%和72.0%，当pH从7增加到11时，标记效率提高了15.5%。网上版载有补充材料，可在10.1007/s00216-021-03587-y查阅。
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is widely used as a crosslinker for fluorescence labeling of protein in the fields of biochemistry and food analysis. Many natural polysaccharides often contain some proteins or peptides that are very low in content but play a vital role in their biological function as well as technical applications. Determination of these low-content proteinaceous matters requires a highly sensitive and selective method. In this study, a methodological approach for investigations of the presence of proteinaceous material over the molar mass distribution (MD) of polysaccharides was developed using gum acacia (GA) as a model polysaccharide. EDC fluorescence-labeling method was modified by changing the pH (7, 9, and 11) of the solution for the analysis of low-content protein in food materials. Fluorescence spectroscopy and asymmetrical flow field-flow fractionation (AF4) were employed for characterizing the labeling efficiency and physiochemical properties of unlabeled and fluorescence-labeled GA. AF4 provided molar mass (M) and the radius of gyration (rG) of arabinogalactan (AG) and arabinogalactan protein complex (AGP) and determined the presence of proteinaceous matter over the MD. The labeling efficiencies of GA at pH 7, 9, and 11 determined by fluorescence spectroscopy were 56.5, 68.4, and 72.0%, respectively, with an increment of 15.5% when pH was increased from 7 to 11. The modified EDC fluorescence-labeling method allows highly sensitive and selective analysis of low-content proteinaceous matters and their distribution in natural polysaccharides. The online version contains supplementary material available at 10.1007/s00216-021-03587-y.
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DOI：
10.1074/mcp.m111.016238
发表时间：
2012-08
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Olsson N;James P;Borrebaeck CA;Wingren C
通讯作者：
Wingren C
影响因子：
4.8
作者：
Bergfeld, Anne K.;Pearce, Oliver M. T.;Varki, Ajit
通讯作者：
Varki, Ajit
影响因子：
--
作者：
Jo SH;Lee SG;Lee YH
通讯作者：
Lee YH
影响因子：
10.7
作者：
Alftren, Johan;Penarrieta, J. Mauricio;Nilsson, Lars
通讯作者：
Nilsson, Lars
影响因子：
3.1
作者：
Andres-Brull, Marina;Al-Assaf, Saphwan;Jackson, Kevin
通讯作者：
Jackson, Kevin