利用SWATH及同位素標記MRM技術分析基因改造與非基因改造黃豆之差異蛋白質體學研究
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2021
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因人口迅速增加、氣候變遷及各個國家的糧食需求，造成全球於糧食作物上的短缺，進而發展出基因改造作物，並因此蓬勃發展。雖基因改造可改善作物種植並減緩缺糧問題，但目前基因改造作物於食品安全上仍存有疑慮。本實驗首先利用SWATH無標記相對定量技術搭配四極桿飛行時間式質譜儀，定量基因改造與非基因改造黃豆蛋白間含量變化。經純化後，黃豆樣品會先經由鹼性逆相層析法(bRP)進行第一維分離，降低樣品複雜度並增加鑑定蛋白質數量，再搭配奈米級液相層析連接串聯式質譜儀進行分析。在此SWATH分析實驗中，總共鑑定到689個蛋白質及8951個不重複的胜肽，且其中339個蛋白質具定量結果。之後利用MRM方法進行定量，根據SWATH分析結果，挑選出5段可用於MRM分析胜肽，進行二甲基同位素標記，在一次實驗中定量分析多種樣品。同位素標記結合多重反應監測的相對定量結果與SWATH結果皆具有相同趨勢，因此可驗證SWATH分析與同位素標記結合多重反應監測方法皆擁有高可信度。最後，篩選出26個差異蛋白質進行生物途徑資料庫GO與STRING分析，發現於基因改造與非基因改造黃豆中，差異蛋白質的生物途徑及功能與核醣體、營養儲存、氧化還原、細胞質及胞質溶膠有相關。本研究為首度使用SWATH與同位素標記結合MRM方法來定量基改與非基改黃豆差異蛋白質體，期望透過差異蛋白質體學研究與生物途徑資料分析增進對基改黃豆作物之瞭解。
In recent years, the rapid population growth, climate change, and large demand from developing countries have caused a global shortage of crops, thus genetically modified crops have been developed as a result. Although genetic modification can improve crop planting and solve food shortages, it is currently impossible to confirm the food safety concerns of genetically modified crops. In this study, SWATH label-free relative quantification method was used to investigate protein profiles in genetically modified and non-genetically modified soybeans. In order to decrease sample complexity, the purified soybean samples were fractionated by basic reverse phase chromatography followed by nano-flow liquid chromatography-tandem mass spectrometry. A total of 689 proteins and 8951 unique peptides were identified, and 339 proteins were quantified by SWATH analysis. The SWATH relative quantitative results were further confirmed with the isotope labeling with multiple reaction monitoring (MRM), and they showed the same trend. Finally, GeneGO and STRING analyses were performed using 26 selected differentially expressed proteins. They are found to be associated with the biological and functions pathways including ribosome, seed storage protein and cytoplasm. To the best of our knowledge, this study is the first report using SWATH and isotope labeling MRM methods for the quantitative proteomics analysis for the GM and non-GM soybeans. We believe this research will be beneficial to the understanding of genetically modified soybean crops and their usage in the future.
In recent years, the rapid population growth, climate change, and large demand from developing countries have caused a global shortage of crops, thus genetically modified crops have been developed as a result. Although genetic modification can improve crop planting and solve food shortages, it is currently impossible to confirm the food safety concerns of genetically modified crops. In this study, SWATH label-free relative quantification method was used to investigate protein profiles in genetically modified and non-genetically modified soybeans. In order to decrease sample complexity, the purified soybean samples were fractionated by basic reverse phase chromatography followed by nano-flow liquid chromatography-tandem mass spectrometry. A total of 689 proteins and 8951 unique peptides were identified, and 339 proteins were quantified by SWATH analysis. The SWATH relative quantitative results were further confirmed with the isotope labeling with multiple reaction monitoring (MRM), and they showed the same trend. Finally, GeneGO and STRING analyses were performed using 26 selected differentially expressed proteins. They are found to be associated with the biological and functions pathways including ribosome, seed storage protein and cytoplasm. To the best of our knowledge, this study is the first report using SWATH and isotope labeling MRM methods for the quantitative proteomics analysis for the GM and non-GM soybeans. We believe this research will be beneficial to the understanding of genetically modified soybean crops and their usage in the future.
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基因改造, 黃豆, 蛋白質體學, 同位素標記, 雙甲基標記, 標靶蛋白質體學, genetically modified, soybean, proteomic, SWATH, isotope label, dimethyl label, MRM