An integrated shotgun proteomics and bioinformatics approach for analysis of brain proteins from MCAO model using serial affinity chromatograph with four active ingredients from Shengmai preparations as ligands

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Identification and validation of disease-relevant target proteins for natural products is an essential component of modern pharmaceutical research. In the present study, an integrated shotgun proteomics and bioinformatics approach was established to profile the interaction of active small molecules derived from ShengMai preparations (SMXZF) with hundreds of endogenously expressed proteins from middle cerebral artery occlusion (MCAO) model. Affinity-based proteomic strategies for isolation and identification of targets for the bioactive components is a classic, but still powerful approach. The proteins bound by SMXZF of the brain tissue proteins from MCAO model via serial affinity chromatograph were analyzed by nano liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) and all MS/MS spectra were then automatically searched by the SEQUEST program. A total of 154 proteins had been identified, with the molecular weight ranging from 21,369.6 to 332,393.21 and the pI from 4.32 to 10.88. Bioinformatic analysis was also implemented to better understand the identified proteins. In the gene ontology (GO) annotation, all the identified proteins were classified into 39, 18 and 12 groups according to biological process, cellular component and molecular function, respectively. KEGG pathways analysis of the identified proteins was conducted with 46 corresponding pathways found. In addition, the gene network was also constructed to analyze the relationship of these genes each other. Further validation of some targets were performed in MCAO model by Western blotting. The results indeed supported the notion that proteins MAPK/ERK1/2, CaMKII and VIM were involved in the disease development of MCAO and played an essential role in the protective effect of SMXZF. This study highlights the effectiveness and reliability of this integrated shotgun proteomics and bioinformatics approach, which is a promising paradigm for target identification and elucidating the mechanism of natural products in future research.

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