Sample preparation methods used for genetically modified organisms (GMOs) analysis are often time consuming, require extensive manual manipulation, and result in limited amounts of purified protein, which may complicate the detection of low-abundance GM protein. A robust sample pretreatment method prior to mass spectrometry (MS) detection of the transgenic protein (5-enolpyruvylshikimate-3-phosphate synthase [CP4 EPSPS]) present in Roundup Ready soya is investigated. Liquid chromatography-multiple reaction monitoring tandem MS (nano LC-MS/MS-MRM) was used for the detection and quantification of CP4 EPSPS. Gold nanoparticles (AuNPs) and concanavalin A (Con A)-immobilized Sepharose 4B were used as selective probes for the separation of the major storage proteins in soybeans. AuNPs that enable the capture of cysteine-containing proteins were used to reduce the complexity of the crude extract of GM soya. Con A-sepharose was used for the affinity capture of β-conglycinin and other glycoproteins of soya prior to enzymatic digestion. The methods enabled the detection of unique peptides of CP4 EPSPS at a level as low as 0.5% of GM soya in MRM mode. Stable-isotope dimethyl labeling was further applied to the quantification of GM soya. Both probes exhibited high selectivity and efficiency for the affinity capture of storage proteins, leading to the quantitative detection at 0.5% GM soya, which is a level below the current European Union's threshold for food labeling. The square correlation coefficients were greater than 0.99. The approach for sample preparation is very simple without the need for time-consuming protein prefractionation or separation procedures and thus presents a significant improvement over existing methods for the analysis of the GM soya protein.

中文翻译：

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质谱联用亲和探针鉴定转基因大豆中的 CP4 EPSPS。


用于转基因生物 (GMO) 分析的样品制备方法通常非常耗时，需要大量的手动操作，并且纯化的蛋白质数量有限，这可能会使低丰度 GM 蛋白质的检测变得复杂。研究了一种在质谱 (MS) 检测农达大豆中存在的转基因蛋白（5-烯醇丙酮莽草酸-3-磷酸合酶 [CP4 EPSPS]）之前的稳健样品预处理方法。采用液相色谱-多反应监测串联 MS（纳诺 LC-MS/MS-MRM）对 CP4 EPSPS 进行检测和定量。金纳米粒子 (AuNP) 和固定刀豆球蛋白 A (Con A) 的 Sepharose 4B 用作选择性探针，用于分离大豆中的主要储存蛋白。 AuNP 能够捕获含半胱氨酸的蛋白质，用于降低转基因大豆粗提物的复杂性。 Con A-琼脂糖用于在酶消化之前亲和捕获大豆中的 β-伴大豆球蛋白和其他糖蛋白。该方法能够在 MRM 模式下检测含量低至转基因大豆 0.5% 的 CP4 EPSPS 独特肽。稳定同位素二甲基标记进一步应用于转基因大豆的定量。两种探针对储存蛋白的亲和捕获都表现出高选择性和高效率，从而可以对 0.5% 转基因大豆进行定量检测，该水平低于当前欧盟食品标签的阈值。平方相关系数大于0.99。样品制备方法非常简单，无需耗时的蛋白质预分级或分离程序，因此比现有的转基因大豆蛋白分析方法有了显着改进。