基于不同沉淀方法检测小鼠心脏组织中S-棕榈酰化蛋白
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摘要
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.
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.
引文
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2 Schmidt M F,Bracha M,Schlesinger M J.Proc.Natl.Acad.Sci.USA,1979,76(4):1687-1691
3 Paige L A,Nadler M J S,Harrison M L,Cassady J M,Geahlen R L.J.Biol.Chem.,1993,268(12):8669-8674
4 Kong E,Peng S,Chandra G,Sarkar C,Zhang Z,Bagh M B,Mukherjee A B.J.Biol.Chem.,2013,288(13):9112-9125
5 FANG Cai-Yun,ZHANG Xiao-Qin,LU Hao-Jie.Chinese J.Anal.Chem.,2014,42(4):616-622方彩云,张晓勤,陆豪杰.分析化学,2014,42(4):616-622
6 Wei X C,Song H W,Semenkovich C F.Arter.Thromb Vasc.Biol.,2014,34(2):346-354
7 Dowal L,Yang W,Freeman M R,Steen H,Flaumenhaft R.e-Blood,2011,118(13):62-73
8 Sanders S S,Martin D D O,Butland S L,Lavallée-Adam M,Calzolari D,Kay C,Yates J R,Hayden M R.PLo S Comput.Biol.,2015,11(8):1-20
9 Pinner A L,Tucholski J,Haroutunian V,Mc Cullumsmith R E,Meador-Woodruff J H.Schizophr.Res.,2016,177(1-3):78-87
10 Wan J M,Savas J N,Roth A F,Sanders S S,Singaraja R R,Hayden M R,Yates J R,Davis N G.Chem.Biol.,2013,20:1421-1434
11 Lanyon-Hogg T,Faronato M,Serwa R A,Tate E W.Trends Biochem.Sci.,2017,42(7):566-581
12 Runkle K B,Kharbanda A,Stypulkowski E,Cao X J,Wang W,Garcia B A,Witze E S.Mol.Cell,2016,62(3):385-396
13 Pei Z,Xiao Y,Meng J,Hudmon A,Cummins T R.Nat.Commun.,2016,7:1-13
14 Zhou T,Li J,Zhao P,Liu H,Jia D,Jia H,He L,Cang Y,Boast S,Chen Y H,Thibault H,Scherrer-Crosbie M,Goff S P,Li B.Proc.Natl.Acad.Sci.USA,2015,112(51):15666-15671
15 WANG Zi-Jian,GAO Xiang,YANG Jing-Bo,SUN Wan-Chun,WU Dong-Lin,PENG Qi-Sheng,LIU Ning.Chinese J.Anal.Chem.,2016,44(10):1521-1527王子健,高祥,杨静波,孙万春,吴东林,彭其胜,刘宁.分析化学,2016,44(10):1521-1527
16 Wan J,Roth A F,Bailey A O,Davis N G.Nat.Protoc.,2007,2(7):1573-1584