"How different are 2D and 3D in vitro model for investigating the mechanism of action of bioactive compounds? The case of phytocannabinoid CBDA"_3D IP DMSO

The scope of the present work concerned the investigation of the CBDA-EIF2A interaction in 3D-U87MG cell model.
For Immunoprecipitation analysis, U87MG cells were cultured in 3D-cell model and then treated with DMSO or CBDA. Once obtained the protein lysate, Co-IP of EIF2A was performed and the samples underwent to in-gel digestion with trypsin enzyme and processed for LC-MS/MS analysis.
Raw files obtained from the QExactive mass spectrometer were processed with Proteome Discoverer software. Trypsin was used as the enzyme for protein digestion. Fasta file UP000005640_9606 was set as protein database. The maximum number of missed cleavages was set to 2. Unique peptides were set for quantification. Precursor and fragment mass tolerance were set, respectively, as 10ppm and 0.02 Da. Quantitative analysis was set as ratio 3D CBDA/3D DMSO.

本研究聚焦于3D-U87MG细胞模型中CBDA与EIF2A的相互作用探究。
免疫沉淀（Immunoprecipitation, IP）分析实验中，将U87MG细胞培养于3D细胞模型，随后分别以二甲基亚砜（Dimethyl Sulfoxide, DMSO）与CBDA进行处理。获取蛋白裂解液后，对EIF2A开展免疫共沉淀（Co-Immunoprecipitation, Co-IP）实验，将所得样品经胰蛋白酶（trypsin）进行胶内酶解，随后采用液相色谱-串联质谱（Liquid Chromatography-Tandem Mass Spectrometry, LC-MS/MS）完成分析。
从QExactive质谱仪（QExactive mass spectrometer）获取的原始数据文件，通过Proteome Discoverer软件（Proteome Discoverer）进行处理。蛋白酶解所用酶为胰蛋白酶，选用的蛋白质数据库为FASTA格式文件UP000005640_9606。最大允许漏切肽段数设置为2，以唯一肽段作为定量分析的依据。前体离子与碎片离子的质量容忍度分别设置为10ppm与0.02Da。定量分析以3D CBDA组与3D DMSO组的信号比值作为计算基准。