Quantitative analysis of TMT phosphorylation modification to investigate the effect of verbascoside on the expression of phosphorylated protein in AD cell model

Background—The active monomer Verbascum glycosides in Cistanche tubulosa has good development prospects in terms of neuroprotection and delaying neurodegenerative diseases, and it has become one of the research hot spots. To investigate the effect of verbascoside (OC1) on the expression of phosphorylated protein in the protective effect of AD cell model by TMT labeling and phosphorylation enrichment technique and high-resolution liquid chromatography-mass spectrometry quantitative proteomics research strategy. Methods—The normal control group, the model group Aβ1-42(10μmol/L) group and the OC1(10μg/ml) administration group were set. (1)protein extraction quality control. (2)TMT mark. (3)HPLC classification and modification enrichment. (4)Analysis of mass spectrometry by liquid chromatography-mass spectrometry. (5)Analysis of bioinformatics results. (6)Western Blotting was used to detect the expression levels of p-CaMKII(Thr286), p-Synapsin1(Ser603)/Synapsin1, Synaptophysin and Synaptotagmin-1 protein. Results—The study finally identified 9020 phosphorylation sites on 3227 proteins, of which 8635 sites of 3134 proteins contained quantitative information. Screening of differential sites follows the following criteria: 1.2 times the change threshold and CV value < 0.1. Based on the above data and standards, we performed a systematic bioinformatics analysis of proteins containing quantitative information sites. Western Blotting results showed that Verbascoside could promote the expression of p-CaMKII (Thr286), p-Synapsin (Ser603)/Synapsin, Synaptophysin and Synaptotagmin-1 protein. Conclusions—Verbascoside(OC1) can increase the expression of phosphorylated protein in AD cell model, which provides a basis for further study on the molecular mechanism of verbascoside promoting neurotransmitter release.