Department of Chemistry, Rajiv Gandhi University of Knowledge Technologies, Hyderabad, India.
Department of Chemistry, National Dong Hwa University, Hualien, 97401, Taiwan.
J Mass Spectrom. 2020 Feb;55(2):e4371. doi: 10.1002/jms.4371. Epub 2019 Jun 18.
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.
用于分析转基因生物（GMO）的样品制备方法通常耗时耗力，需要大量的手动操作，并且得到的纯化蛋白量有限，这可能会使低丰度 GM 蛋白的检测复杂化。研究了一种在质谱（MS）检测抗农达大豆中存在的转基因蛋白（5-烯醇丙酮酰莽草酸-3-磷酸合酶[CP4 EPSPS]）之前进行强大的样品预处理方法。使用液相色谱-多重反应监测串联质谱（nano LC-MS/MS-MRM）检测和定量 CP4 EPSPS。金纳米粒子（AuNPs）和伴刀豆球蛋白 A（Con A）固定化琼脂糖 4B 被用作分离大豆中主要储存蛋白的选择性探针。用于捕获含半胱氨酸蛋白的 AuNPs 用于减少 GM 大豆粗提物的复杂性。Con A-琼脂糖用于在酶解之前亲和捕获β-伴大豆球蛋白和其他大豆糖蛋白。该方法能够以 MRM 模式检测 CP4 EPSPS 的独特肽，其水平低至 GM 大豆的 0.5%。进一步将稳定同位素二甲基标记应用于 GM 大豆的定量。两种探针均表现出对储存蛋白的亲和捕获的高选择性和效率，导致在 0.5% GM 大豆的定量检测，这低于欧盟目前对食品标签的阈值。方相关系数大于 0.99。该样品制备方法非常简单，不需要耗时的蛋白质预分级或分离程序，因此与现有的 GM 大豆蛋白分析方法相比有了显著的改进。
