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Citation: CHENG Wei, ZHAN Fang-Ling, LI Shui-Ming, YANG Jing-Bo, WANG Yong, LIU Ning. Detection of S-palmitoylated Proteins in Mouse Heart Tissue Based on Different Precipitation Methods[J]. Chinese Journal of Analytical Chemistry, ;2019, 47(1): 30-37. doi: 10.19756/j.issn.0253-3820.181619 shu

Detection of S-palmitoylated Proteins in Mouse Heart Tissue Based on Different Precipitation Methods

1.
Central Laboratory, the Second Hospital of Jilin University, Key Laboratory of Zoonosis Research, Ministry of Education, Changchun 130041, China;
2.
College of Life and Ocean Science, Shenzhen University, Shenzhen 518060, China

Received Date: 28 September 2018
Revised Date: 19 November 2018

Fund Project: This work was supported by the National Natural Science Foundation of China (No.81472030) and the Jilin Province Science and Technology Department (No.20180101267JC)

The acyl-biotinyl exchange (ABE) method is a common method for detection of S-palmitoylated proteins by replacing palmitic acid with biotin. In this study, we compared the effects of acetone precipitation and methanol-chloroform precipitation on the detection of S-palmitoylation protein in acyl-biotin exchange method, and analyzed the S-palmitoylated protein in mouse cardiac tissue. First, N-ethylmaleimide (NEM) was used to block the free sulfhydryl on the protein molecules, and then biotinylation reagent (HPDP-Biotin) was used to label the new produced cysteine thiol cleaved by hydroxylamine (HA) in mouse heart tissue. During the ABE reaction, excess unreacted NEM, HA, and HPDP-Biotin were removed by precipitation of the protein. The S-palmitoylated protein in total protein of heart tissue was labeled with ABE reaction based on different precipitation methods, and the S-palmitoylated protein labeled with biotin was enriched by streptavidin agarose beads. The enriched proteins were analyzed by mass spectrometry, and 50 S-palmitoylated proteins were identified. The acetone precipitation assay group identified 23 S-palmitoylated proteins, and the methanol-chloroform precipitation experimental assay group identified 37 S-palmitoylated proteins. 10 proteins were identified in both groups, and 14 newly identified palmitoylated proteins. The result showed that the cross-complementary combination use of different precipitation methods could be helpful for the identification of palmitoylated proteins.
- Heart,
- S-Palmitoylated protein,
- Precipitation,
- Mass spectrometry
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