基于不同沉淀方法检测小鼠心脏组织中S-棕榈酰化蛋白

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

酰基-生物素置换法(Acyl-biotinyl exchange,ABE)是利用生物素取代棕榈酸,进而对棕榈酸修饰的蛋白进行标记检测的方法,是检测S-棕榈酰化蛋白质的常用方法。本研究比较了ABE法中丙酮沉淀与甲醇-氯仿沉淀两种方法对S-棕榈酰化蛋白检测的影响,并对小鼠心脏组织中S-棕榈酰化修饰蛋白进行整体分析,进一步丰富S-棕榈酰化蛋白质的相关研究。首先用N-乙基马来酰亚胺(NEM)对小鼠心脏组织中蛋白质分子上的游离巯基进行封闭,再利用巯基反应性生物素化试剂(HPDP-Biotin)对羟胺(HA)切割后新产生的半胱氨酸巯基进行生物素标记,可检测出小鼠心脏组织中S-棕榈酰化修饰的蛋白。在此反应过程中,均通过蛋白沉淀法除去未反应的NEM、HA及HPDP-Biotin。基于不同沉淀方法对小鼠心脏组织总蛋白中的S-棕榈酰化蛋白进行标记后,利用链霉亲和素修饰琼脂糖珠对已标记上生物素的S-棕榈酰化蛋白进行富集,利用质谱检测分析。本研究共鉴定出50种S-棕榈酰化蛋白,其中丙酮沉淀实验组鉴定出23种S-棕榈酰化蛋白,甲醇-氯仿沉淀实验组鉴定出37种S-棕榈酰化蛋白,两组均鉴定出的蛋白有10种,不同的沉淀方法交叉互补使用可使鉴定结果更加丰富、可靠。

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.