Detection of S-palmitoylated Proteins in Mouse Heart Tissue Based on Different Precipitation Methods
| Autor: | Wei Cheng, Shui-Ming Li, Fang-Ling Zhan, Yong Wang, Jing-Bo Yang, Ning Liu |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Precipitation (chemistry)
010401 analytical chemistry technology industry and agriculture 02 engineering and technology 021001 nanoscience & nanotechnology Mass spectrometry 01 natural sciences 0104 chemical sciences Analytical Chemistry Palmitic acid chemistry.chemical_compound Hydroxylamine Biotin chemistry Biochemistry Reagent Biotinylation lipids (amino acids peptides and proteins) 0210 nano-technology Cysteine |
| Zdroj: | Chinese Journal of Analytical Chemistry. 47:30-37 |
| ISSN: | 1872-2040 |
| DOI: | 10.1016/s1872-2040(18)61134-x |
| Popis: | The acyl-biotinyl exchange (ABE) is widely used for detection of S-palmitoylated proteins by replacing palmitic acid with biotin, which is a common method for detecting S-palmitoylated proteins. In this study, the effects of acetone precipitation and methanol-chloroform precipitation on the detection of S-palmitoylation proteins in acyl-biotin exchange method were compared, and the S-palmitoylated proteins in mouse cardiac tissue were analyzed. First, N-ethylmaleimide (NEM) was used to block free sulfhydryls within protein molecules. Then, biotinylation reagent (HPDP-Biotin) was used to label the newly produced cysteine thiols that were resulted from treatment by hydroxylamine (HA) in mouse heart tissue. During the ABE reaction, excess unreacted NEM, HA and HPDP-Biotin were removed by precipitation of the proteins. Then, the S-palmitoylated proteins from heart tissue were labeled with ABE reaction based on different precipitation methods, and the S-palmitoylated proteins labeled with biotin were enriched by streptavidin agarose beads. The enriched proteins were analyzed by mass spectrometry, and 50 S-palmitoylated proteins were identified. Specifically, 23 S-palmitoylated proteins were identified in acetone precipitation assay group, and 37 S-palmitoylated proteins in the methanol-chloroform precipitation assay group were identified. 10 palmitoylated proteins were identified in both groups. The results showed that the combination of different precipitation methods could be helpful for the identification of palmitoylated proteins. |
| Databáze: | OpenAIRE |
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