基于质谱检测转基因生物外源蛋白质的消化稳定性

摘要

基于无标记定量质谱检测技术建立了一种新的转基因生物外源蛋白质消化稳定性评价方法。以牛β-乳球蛋白、牛血清白蛋白和大豆胰蛋白酶抑制剂为标准蛋白质，经模拟人体胃/肠消化液消化后进行凝胶电泳分离，切取目标蛋白质条带进行酶切，提取肽段进行纳升液相色谱-电喷雾串联质谱分析。基于Mascot数据库检索鉴定目标蛋白质。计算各消化时间点蛋白质匹配肽段数与消化前蛋白质匹配肽段数的比值，当比值≤0.50时判断为目标蛋白质在该时间段内已消化。将此方法应用于转基因抗虫水稻“华恢1号”外源蛋白Cry1Ab/1Ac的消化稳定性分析，结果显示在模拟胃液中消化2 min时，比值下降到0.50以下，在模拟肠液中消化15 s后比值下降到0.50以下，表明该蛋白在胃/肠消化液中具有消化不稳定性。%Digestive stability analysis of exogenous protein is one of the important indexes of geneticaly modified organisms (GMO) safety assessment. In the present study, we established a novel assessment assay for protein digestive stability based on label-free quantification of mass spectrometry using bovineβ-lacto globulin (BLG), bovine serum albumin (BSA) and soybean trypsin inhibitor (STI) as standard proteins. Protein samples were treated with simulated gastric/intestinal fluids (SGF/SIF), and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The target bands were then cut out and trypsinized. The extracted peptides were analyzed by nano liquid chromatography-electronic spray ion-mass spectrum/mass spectrum and identified by Mascot software. By calculating the ratio between the matched peptide numbers of the target protein before and after SGF/SIF treatment, the digestibility of the target protein was estimated. When the ratio was lower than 0.50, the target protein was considered digestible. This newly developed assay was applied to Cry1Ab/1Ac, the exogenous protein of the geneticaly modified rice‘Huahui No. 1’. The results showed that the ratio was lower than 0.50 after digestion in SGF for 2 minutes, while it decreased to less than 0.50 after digestion in SIF for 15 seconds, indicating that it is very liable to be digested. Our study provides a novel MS-based method for digestive stability analysis of target proteins from genetically modified organisms, which is easy to operate with no need for specific antibody.