CN114736238A - 一种含稳定同位素的蛋白质羧基磷酸化标记试剂及其制备方法与应用 - Google Patents
一种含稳定同位素的蛋白质羧基磷酸化标记试剂及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种含稳定同位素的蛋白质羧基磷酸化标记试剂及其制备方法与应用,所述蛋白质羧基磷酸化标记试剂为含氘‑2、碳‑13和氧‑18等稳定同位素标记的有机磷试剂。所述标记试剂的制备步骤为:(1)叔丁氧羰基保护的溴胺的制备;(2)叔丁氧羰基保护的含稳定同位素的氨基膦酸酯的制备:(3)含稳定同位素的氨基膦酸酯的制备。本发明所提供的标记试剂可作为蛋白质羧基磷酸化修饰标记分子探针,对蛋白质羧基磷酸化修饰位点特有的羧基磷酸混酐基团进行选择性高效标记,实现具有不稳定性的羧基磷酸化修饰到稳定酰胺结构的化学转换,然后通过质谱表征进行定性和定量分析。本发明所提供的方法具有较好的准确性、高灵敏度及广泛的适用性等优点。
Description
技术领域
本发明属于蛋白标记试剂领域,具体涉及一种含稳定同位素的蛋白质羧基磷酸化标记试剂及其制备方法与应用。
背景技术
蛋白质可逆磷酸化是生命过程中最重要的翻译后修饰之一,几乎参与了全部的生命调控过程,例如:细胞周期调控、信号转导、分化、程序性死亡和代谢等1。编码蛋白激酶和磷酸酶的基因大约占据真核生物基因组的2%-5%,其中人类基因组中约有100个磷酸酶基因和500个激酶基因2。可逆磷酸化修饰通过蛋白激酶和磷酸酶协同调控,实现对磷酸化底物的磷酸化和去磷酸化;磷酸化底物种磷酸基团的引入可以改变蛋白质的性质,例如蛋白构效、酶活性及蛋白-蛋白相互作用等。蛋白磷酸化修饰往往作为蛋白活性激活和失活调控的重要开关,而可逆磷酸化平衡的失控往往导致严重的疾病,例如癌症、神经退行性疾病等人类重大疾病的发生3。
根据磷酸化氨基酸残基的不同,可以将磷酸化蛋白质分为四类:第一类为O磷酸酯修饰,通过氨基酸侧链羟基的磷酸化形成,如Ser、Thr和Tyr以及羟脯氨酸和羟赖氨酸4;第二类为N磷酸酯修饰5,通过氨基酸侧链氨基的磷酸化形成,如Lys、Arg和His;第三类为S磷酸酯修饰,通过Cys的磷酸化形成;第四类为羧基混酐修饰,通过Glu和Asp的侧链羧基磷酸化形成6。其中第一类磷酸化修饰最为稳定,研究也最为深入,多种技术可以实现高灵敏检测,例如高特异性抗体、纳米富集技术及组学质谱技术7等;第二类和第三类磷酸化修饰近年来得到快速发展,特别是组氨酸N-磷酸化修饰激酶和酯酶的发现和机制研究。羧基磷酸化修饰在生命过程中发挥着关键作用,多种酶设计该磷酸化修饰,例如各种生物细胞质膜中的Ca2+ATP酶、H+ATP酶和Na+/K+ATP酶等离子泵的P-型ATP酶8-10,另外还有丝氨酸磷酸化酶等磷酸酶11、β-葡萄糖磷酸变位酶等磷酸变位酶以及磷酸乙醛水解酶12-14和微生物双组分系统15等。然而,第四类磷酸化修饰虽然发现很早,但发展缓慢,主要原因是羧基磷酸化修饰具有羧基-磷酸混酐结构,酸碱条件下具有化学和热不稳定性,十分容易水解,因此很难对其进行系统发现和动态过程的定量检测,这极大限制了蛋白质天冬氨酸和谷氨酸侧链羧基磷酸化修饰的发现和功能研究16。
目前已有的研究针对蛋白质酶解肽段的N端和赖氨酸侧链氨基进行磷试剂标记,具有1-3个数量级的质谱增敏效应,改善肽段的色谱分离及简化测序图谱等多种优势,实现了蛋白质高灵敏检测分析17,18。因此,通过设计稳定同位素标记氨基膦酸酯试剂,靶向蛋白质种羧基磷酸化修饰位点,把不稳定的羧基-磷酸混酐结构转化为化学稳定的酰胺结构,并应用高分辨质谱技术对羧基磷酸化修饰位点进行鉴定,建立羧基磷酸化修饰的高效检测和定量分析新技术,对羧基磷酸化修饰的系统发现和生物功能揭示具有重要意义。
主要参考文献
1.Thorner,J.;Hunter,T.;Cantley,L.C.;Sever,R.,Signal transduction:Fromthe atomic age to the post-genomic era.Cold Spring Harb Perspect Biol 2014,6(12),a022913.
2.Cohen,P.,The development and therapeutic potential of proteinkinase inhibitors.Curr Opin Chem Biol 1999,3(4),459-65.
3.Cohen,P.,The role of protein phosphorylation in human health anddisease.The Sir Hans Krebs Medal Lecture.Eur J Biochem 2001,268(19),5001-10.
4.Wang,R.;Wang,G.,Protein Modification and AutophagyActivation.Advances in experimental medicine and biology 2019,1206,237-259.
5.Hu,Y.;Jiang,B.;Zhang,L.;Zhang,Y.,Advancesin enrichment anddetection methods for N-phosphorylated proteins.Se Pu 2020,38(3),278-286.
6.Marmelstein,A.M.;Moreno,J.;Fiedler,D.,Chemical Approaches toStudying Labile Amino Acid Phosphorylation.Topics in current chemistry(Cham)2017,375(2),22.
7.Areces,L.B.;Matafora,V.;Bachi,A.,Analysis of proteinphosphorylation by mass spectrometry.European journal of mass spectrometry(Chichester,England)2004,10(3),383-92.
8.Scarborough,G.A.,Structure and function of the P-type ATPases.CurrOpin Cell Biol 1999,11(4),517-22.
9.Jorgensen,P.L.;Pedersen,J.,Role of Conserved TGDGVND-Loop in Mg2+Binding,Phosphorylation,and Energy Transfer in Na,K-ATPase.Journal ofBioenergetics&Biomembranes 2001.
10.Weidemuller,C.;Hauser,K.,Ion transport and energy transduction ofP-type ATPases:implications from electrostatic calculations.Biochim BiophysActa 2009,1787(6),721-9.
11.Collet,J.F.;Stroobant,V.;Van Schaftingen,E.,Mechanistic studies ofphosphoserine phosphatase,an enzyme related to P-type ATPases.J Biol Chem1999,274(48),33985-90.
12.Allen,K.N.;Dunaway-Mariano,D.,Phosphoryl group transfer:evolutionof a catalytic scaffold.Trends Biochem Sci 2004,29(9),495-503.
13.Morais,M.C.;Zhang,G.;Zhang,W.;Olsen,D.B.;Dunaway-Mariano,D.;Allen,K.N.,X-ray crystallographic and site-directed mutagenesis analysis of themechanism of Schiff-base formation in phosphonoacetaldehyde hydrolasecatalysis.J Biol Chem 2004,279(10),9353-61.
14.Borys;Szefczyk,Towards understanding phosphonoacetaldehydehydrolase:an alternative mechanism involving proton transfer that triggers P-C bond cleavage.Chemical communications(Cambridge,England)2008.
15.Hess,J.F.;Oosawa,K.;Kaplan,N.;Simon,M.I.,Phosphorylation of threeproteins in the signaling pathway of bacterial chemotaxis.Cell 1988,53(1),79-87.
16.Hauser,A.;Penkert,M.;Hackenberger,C.P.R.,Chemical Approaches toInvestigate Labile Peptide and Protein Phosphorylation.Accounts of ChemicalResearch 2017,50(8),1883-1893.
17.Zhang,S.;Shi,J.;Shan,C.;Huang,C.;Wu,Y.;Ding,R.;Xue,Y.;Liu,W.;Zhou,Q.;Zhao,Y.;Xu,P.;Gao,X.,Stable isotope N-phosphoryl amino acids labeling forquantitative profiling of amine-containing metabolites using liquidchromatography mass spectrometry.Anal.Chim.Acta 2017,978,24-34.
18.Gao,X.;Wu,H.;Lee,K.C.;Liu,H.;Zhao,Y.;Cai,Z.;Jiang,Y.,Stableisotope N-phosphorylation labeling for Peptide de novo sequencing and proteinquantification based on organic phosphorus chemistry.Anal Chem 2012,84(23),10236-44.
发明内容
针对上述现有技术存在的不足之处,本发明目的在于提供一种含稳定同位素的蛋白质羧基磷酸化标记试剂及其制备方法与应用。为了达成上述目的,本发明的解决方案是:
一种含稳定同位素的蛋白质羧基磷酸化标记试剂,其特征在于其结构式如下:
结构中:a为1或2;b为12或13;c为16或18;n为1、2、3、4或5;n1为1、2、3、4、5、6、7、8、9或10。
所述含稳定同位素的蛋白质羧基磷酸化标记试剂的制备方法,其特征在于包括以下步骤:
(1)叔丁氧羰基保护的溴胺的制备:低温下将二碳酸二叔丁酯、溴胺化合物置于有机溶剂中,逐滴加入有机碱后将反应混合物升温到室温继续搅拌;然后除去溶剂,将所得剩余物纯化后即可制得叔丁氧羰基保护的溴铵;
(2)叔丁氧羰基保护的含稳定同位素的氨基膦酸酯的制备:将钠氢悬浮在无水有机溶剂中,然后逐滴加入含稳定同位素的亚磷酸酯,将所得混合物在室温下反应后加热回流,接下来在低温下加入步骤(1)中制备的叔丁氧羰基保护的溴铵,将所得混合物在室温下继续反应,最后用水淬灭反应,将淬灭后所得产物纯化后即可制得叔丁氧羰基保护的含稳定同位素的氨基膦酸酯;
(3)含稳定同位素的氨基膦酸酯的制备:将步骤(2)制备的叔丁氧羰基保护的含稳定同位素氨基膦酸酯溶于有机溶剂后加入酸,所得反应混合物在室温下搅拌反应,除去溶剂后将所得反应剩余物纯化后即可制得含稳定同位素的氨基膦酸酯。
优选地,步骤(1)和步骤(3)中所述的有机溶剂选自乙酸乙酯,氯仿或二氯甲烷中的一种。
优选地,所述的低温均为冰浴条件。
优选地,步骤(1)中所述的溴胺化合物为溴甲胺,2-溴乙胺,3-溴丙胺,4-溴丁胺,5-溴戊胺或6-溴己胺中的一种;所述的有机碱为吡啶、二乙胺,二异丙胺或三乙胺中的一种;所述的室温搅拌反应的时间为16-24小时。
优选地,步骤(2)中所述的无水有机溶剂为无水N,N-二甲基甲酰胺,无水1,4-二氧六环或无水四氢呋喃中的一种。
优选地,步骤(2)中所述的含稳定同位素的亚磷酸酯为亚磷酸二甲酯,亚磷酸二乙酯,亚磷酸二丙酯,亚磷酸二丁酯或亚磷酸二戊酯中的一种。
优选地,步骤(3)中所述的酸为盐酸,甲酸,醋酸或三氟乙酸中的一种。
所述的含稳定同位素的蛋白质羧基磷酸化标记试剂在鉴定蛋白质羧基磷酸化修饰中的应用,其特征在于具体步骤如下:
(1)蛋白乙酰磷酸化反应:将蛋白质加入到乙酰磷酸化试剂中进行恒温反应;
(2)标记反应:将步骤(1)中经过乙酰磷酸处理过的蛋白质与缓冲溶液和标记试剂一起进行标记反应获得标记蛋白产物;其中,所用的缓冲溶液为碱性缓冲溶液;标记反应温度为0℃至室温;标记反应的时间为0.5h-1h;
(3)LC-MS/MS检测:将步骤(2)中获得的标记蛋白产物除盐后进行LC-MS/MS检测,记录LC-MS/MS谱图;
色谱条件如下:采用超高压纳升液相色谱;流速为300nL/min;A相为水溶液,B相为ACN溶液;
质谱参数如下:采用纳升级ESI离子源,正离子模式,喷雾电压为3.5KV,毛细管温度为360℃;采用飞行时间或轨道离子阱(orbitrap)质量分析器,质量扫描范围m/z 350-1800;
(4)蛋白组学数据分析:将步骤(3)中获得的LC-MS/MS谱图进行检索与序列比对分析;其中,在序列比对过程中需加入目的蛋白序列库作为数据库,同时还需要添加不同的蛋白固定修饰和动态修饰。
优选地,步骤(1)中所述的恒温反应温度为37℃,所述的恒温反应时间为1h。
优选地,步骤(2)中所述的碱性缓冲溶液为四乙基溴化铵溶液。
优选地,步骤(2)中所述的标记反应中标记试剂和蛋白的浓度比例为10-1000:1。
本发明的具体原理如下:
本发明所提供的含稳定同位素的蛋白质羧基磷酸化标记试剂可作为蛋白质羧基磷酸化标记分子探针对蛋白质羧基磷酸化修饰位点特有的羧基磷酸混酐基团进行选择性高效标记,然后通过质谱分析对羧基磷酸化修饰进行定性和定量分析。
这里以AEP(β-氨基膦酸酯)试剂在鉴定蛋白质磷酸化天冬氨酸和谷氨酸位点中的应用为例进行原理说明,具体如图2所示:β-氨基膦酸二乙酯分子探针能够特异性识别磷酸化天冬氨酸和磷酸化谷氨酸的羧基磷酸化位点,将不稳定的酰基磷酸酐结构转变为稳定的酰胺结构,从而可以通过质谱对羧基磷酸化位点进行检测和识别。
更进一步,以β-氨基膦酸酯(AEP)两标试剂在定量蛋白质磷酸化位点中的应用为例进行原理说明,具体如图3所示:图3a为AEP-两标结构示意图,图3b为鉴定反应调节蛋白上磷酸化位点策略图。从图3中可以看出:将分子量相差4Da的AEP-两标试剂分别加入到两个不同的蛋白质羧基磷酸化修饰样品中,AEP试剂可以和蛋白质羧基磷酸化发生特异性反应形成稳定的酰胺化学键结构,将含有不同质量标签的AEP试剂添加到羧基磷酸化蛋白样品上,通过LC-MS/MS分析就可以定量分析不同样品中蛋白质羧基磷酸化修饰的差异变化。
本发明的优点:
(1)本发明所提供的含稳定同位素的蛋白质羧基磷酸化标记试剂的制备方法具有所需原料廉价易得、反应条件温和、标记效率高、纯化方法简单及产率高的优点。
(2)本发明提供了蛋白质不稳定磷酸化修饰中羧基磷酸化修饰的鉴定方法,通过磷试剂标记实现了不稳定结构到稳定酰胺结构的官能团转化,分析结构不会受到样品制备过程的影响,排除干扰因素,提高了羧基磷酸化修饰分析的准确性和重复性。
(3)本发明所提供的含稳定同位素的蛋白质羧基磷酸化标记试剂可成功鉴定出KdpE中52位天冬氨酸的羧基磷酸化修饰位点。
(4)本发明所提供的含稳定同位素的蛋白质羧基磷酸化标记试剂可成功鉴定出VicR中52位天冬氨酸的羧基磷酸化修饰位点。
(5)本发明所提供的含稳定同位素的蛋白质羧基磷酸化标记试剂可成功鉴定出体外激酶反应中VicR天冬氨酸的羧基磷酸化修饰位点。
附图说明
图1为含稳定同位素的蛋白质羧基磷酸化标记磷试剂的结构式图。
图2为AEP(β-氨基膦酸酯)试剂在鉴定蛋白质磷酸化天冬氨酸和谷氨酸位点中的应用原理图。
图3为定量分析蛋白质羧基磷酸化位点示意图。
图4为乙酰磷酸(AcP)处理得到pKdpE的标记实验结果图,其中:a为标记实验组和空白对照组的Phos-tag SDS-PAGE结果;b为pKdpE质谱检测的肽段覆盖结果;c为52位天冬氨酸所在肽段磷酸化前后的一级质谱结果;d为52位天冬氨酸所在肽段磷酸化前后的二级裂解质谱结果。
图5为乙酰磷酸(AcP)处理得到pVicR的标记实验结果图;其中:a为pVicR质谱检测的肽段覆盖结果;b为52位天冬氨酸所在肽段磷酸化前后的一级质谱结果;c为52位天冬氨酸所在肽段磷酸化前后的二级裂解质谱结果。
图6为激酶反应所得pVicR的标记实验结果;其中:a为激酶反应所得pVicR质谱检测的肽段覆盖结果;b为192位天冬氨酸所在肽段磷酸化前后的一级质谱结果;c为52位天冬氨酸所在肽段磷酸化前后的二级裂解质谱结果;d为192位天冬氨酸所在肽段磷酸化前后的二级裂解质谱结果。
具体实施方式
下面进一步结合实施例以详细说明本发明。同样应理解,以下实施例只用于对本发明进行进一步说明,不能理解为对本发明保护范围的限制,示例中具体的质量、反应时间和温度、工艺参数等也仅是合适范围中的一个示例,本领域的技术人员根据本发明的上述内容做出的一些非本质的改进和调整均属于本发明的保护范围。
如图1所示为本发明所制备的含稳定同位素的蛋白质羧基磷酸化标记试剂的结构式图,结构中:a为1或2;b为12或13;c为16或18;n为1、2、3、4或5;n1为1、2、3、4、5、6、7、8、9或10。
实施例1
含稳定同位素的β-氨基膦酸酯的制备,具体步骤如下:
(1)叔丁氧羰基保护的2-溴乙胺N-Boc-2-bromoethamine合成:
在0℃冰浴条件下,将二碳酸二叔丁酯(2.44mmol,533mg)溶于二氯甲烷(13mL)中,与2-溴乙胺氢溴酸盐(2.71mmol,556mg)一起置于50mL干燥的圆底烧瓶中;然后逐滴加入三乙胺(3.66mmol,508μL),滴加时间超过10min。滴加完毕将反应混合物升温到室温并继续搅拌反应18h。反应结束后加入二氯甲烷(20mL)稀释反应液,并依次用饱和氯化铵溶液(20mL)、饱和碳酸氢钠溶液(20mL)和饱和氯化钠溶液(20mL)分别洗涤两次。将所得到的有机层溶液用无水硫酸镁干燥,经过滤浓缩后得到最终黄色油状产物,记为N-Boc-2-bromoethamine;
(2)叔丁氧羰基保护的含稳定同位素的β-氨基膦酸酯合成:
将60%钠氢(分散在矿物油中)(2.6mmol,104mg)悬浮在无水四氢呋喃中(10mL),向其中逐滴加入亚磷酸二乙酯(2.6mmol,358.9mg),将所得混合物在常温下反应30min后升温至70℃并加热回流2.5h,然后在0℃条件下加入步骤(1)中制备的N-Boc-2-bromoethamine(2mmol,446.04mg),所得混合物在室温状态反应过夜。反应结束后加入5mL水淬灭反应,水相用二氯甲烷(每次用量10mL)萃取三次后,收集有机相并用无水硫酸镁干燥,经过滤浓缩后得到粗产物。再以硅胶作为固定相,石油醚和乙酸乙酯(v:v=1:1)作为流动相,利用柱层析分离得到黄色油状产物,记为N-Boc-diethyl-AEP;
(3)含稳定同位素的β-氨基膦酸酯合成
将步骤(2)中制备的N-Boc-diethyl-AEP(0.21mmol,9.8mg)溶于二氯甲烷(10mL)后加入三氟乙酸(2.5mL),所得混合物常温下搅拌反应3h后减压蒸馏除去溶剂。将所得三氟乙酸盐用水溶解后加入1M NaOH调节pH至8-9,用二氯甲烷萃取三次,收集有机相加入无水硫酸镁干燥,经过滤浓缩后得到黄色油状产物,记为Diethyl-AEP,结构如下图所示:
Diethyl-AEP(C6H17NO3P):1H NMR(600MHz,CDCl3):δ=4.11-3.99(m,4H),3.02-2.93(m,2H),1.90(dt,J=17.8,6.8Hz,2H),1.26(t,J=7.0Hz,6H)ppm.13C NMR(151MHz,CDCl3):δ=60.67(d,J=6.5Hz),39.58,35.12,15.43(d,J=6.0Hz)ppm.31P NMR(243MHz,CDCl3):δ=30.17ppm.ESI-MS:[M+H]+,m/z 182.0946(理论值182.0929,相对误差为9.3ppm)。
同理,可先由N-Boc-2-bromoethamine和稳定同位素标记的氘代亚磷酸二乙酯4D-DEPH反应得到N-Boc-4D-diethyl-AEP,然后再进一步反应得到4D-diethyl-AEP,其结构如下图所示:
4D-diethyl-AEP(C6H12D4NO3P):1H NMR(600MHz,CDCl3):δ=3.01(s,2H),2.08-1.94(m,2H),1.32(s,6H)ppm.13C NMR(151MHz,CDCl3):δ=61.73-60.79,53.43,35.86,16.18(d,J=5.9Hz)ppm.31P NMR(243MHz,CDCl3):δ=29.76ppm.ESI-MS:[M+H]+,m/z 186.1189(理论值186.1197,相对误差为4.3ppm)。
同理,可先由N-Boc-2-bromoethamine和亚磷酸二异丙酯反应得到N-Boc-diisopropal-AEP,然后进一步反应得到Diisopropal-AEP,其结构如下图所示:
Diisopropal-AEP(C8H20NO3P):1H NMR(600MHz,CDCl3):δ=4.63(ddt,J=12.4,7.9,6.2Hz,2H),2.92(dt,J=16.9,7.0Hz,2H),1.82(dt,J=18.0,7.0Hz,2H),1.25(d,J=6.2Hz,12H)ppm.13C NMR(151MHz,CDCl3):δ=70.10(d,J=6.7Hz),36.47(d,J=4.6Hz),31.25(d,J=139.4Hz),24.06(t,J=4.4Hz)ppm.31P NMR(243MHz,CDCl3):δ=28.28ppm.ESI-MS:[M+H]+,m/z 210.1251(理论值210.1259,相对误差为3.8ppm)。
同理,可先由N-Boc-2-bromoethamine和亚磷酸二甲酯反应得到N-Boc-dimethyl-AEP然后再进一步反应得到Dimethyl-AEP,其结构如下图所示:
Dimethyl-AEP(C4H12NO3P):1H NMR(600MHz,CDCl3):δ=3.67(d,J=11.0Hz,6H),1.40(t,J=12.1Hz,2H),1.18(m,2H)ppm.13C NMR(151MHz,CDCl3):δ=52.24(d,J=6.3Hz),40.62,29.69ppm.31P NMR(243MHz,CDCl3):δ=33.28ppm.ESI-MS:[M+H]+,m/z 154.0626(理论值154.0633,相对误差为4.5ppm)。
同理,可以制备γ-氨基磷酸二甲酯,结构如下式所示:
γ-氨基磷酸二甲酯(C5H14NO3P):1H NMR(600MHz,CDCl3):δ=3.65(d,J=11.0Hz,6H),2.68(t,J=12.1Hz,2H),1.75(m,4H)ppm.13C NMR(151MHz,CDCl3):δ=53.40(d,J=6.0Hz),43.32,28.43,25.31ppm.31P NMR(243MHz,CDCl3):δ=33.37ppm.ESI-MS:[M+H]+,m/z 168.0790。
同理,可以制备ε-氨基磷酸二甲酯,结构如下式所示:
ε-氨基磷酸二甲酯(C7H18NO3P):1H NMR(600MHz,CDCl3):δ=3.65(d,J=11.0Hz,6H),2.69(t,J=12.1Hz,2H),1.75(m,2H),1.52(m,2H),1.25(m,4H)ppm.13C NMR(151MHz,CDCl3):δ=53.40(d,J=6.0Hz),42.31,32.40,31.30,28.30,23.90ppm.31P NMR(243MHz,CDCl3):δ=33.30ppm.ESI-MS:[M+H]+,m/z 196.1103。
实施例2使用Diethyl-AEP分析大肠杆菌蛋白组中乙酰磷酸(AcP)处理后KdpE天冬氨酸羧基磷酸化位点
蛋白质表达和纯化的相关实验步骤如下:
1.大肠杆菌感受态细胞制备步骤如下:
(1)在超净工作台上,用灭过菌的牙签挑取BL21感受态细胞菌种在无抗性的2YT固体培养基平板上划线接菌,于37℃恒温培养箱中培养过夜;
(2)培养过夜后,在平板上挑取三个细菌单菌落,分别接种于5mL 2YT液体培养基中,并在37℃恒温环境及220rpm震荡下培养过夜;
(3)将1mL新鲜过夜培养的大肠杆菌菌液接入到100mL 2YT液体培养基中,置于18℃下的150rpm低温摇床中震荡培养约16h,直到菌液的OD600值达到0.3-0.5之间时停止培养;
(4)将菌液置于冰上冷却10min使其停止生长,再将菌液转移到50ml离心管中,在4℃低温下以2500g转速离心10min后收集菌体,在超净工作台内倒掉上清液并用移液枪尽可能将残余培养基吸尽;
(5)然后加入15mL预冷过的感受态转化缓冲液,在冰上转圈使菌体重悬后,在4℃的2,500g离心10min后在超净工作台内倒掉上清并用移液枪将将剩余液体吸尽,再次加入10mL预冷过的感受态转化缓冲液和700μL DMSO液体,冰上转圈再次将菌体轻柔重悬。
(6)在冰上,将悬浮混匀好的大肠杆菌感受态细胞按50-100μL每管的量迅速分装到预冷的1.5mL离心管中,并立即倒入在液氮罐中预冷的冻存盒中速冻,保存于-80℃。
2.质粒的转化步骤如下:
(1)将感受态细胞从-80℃冰箱中取出,置于冰上融化;
(2)将2μL DNA连接产物或者质粒加入到50μL感受态细胞中,用移液枪反复吹打几次使其混匀后在冰上放置20-30min;
(3)将感受态细胞放入42℃水浴锅内热激45s后迅速拿出放回到冰上静置2-3min;
(4)加入200μL 2YT液体培养基到感受态细胞中,将感受态转化混合液均匀涂布在含有相同抗性的2YT固体培养板上,做上标记后倒置2YT平板于37℃培养箱内过夜培养。
3.重组蛋白表达步骤如下:
(1)将测序正确的质粒转化到感受态细胞BL21中,37℃过夜培养后挑取细菌单菌落,接种于500mL 2YT液体培养基中,在37℃培养至菌液的OD600值达到0.6;
(2)往500mL菌液中加入1mM IPTG诱导重组蛋白的表达,在18℃条件下诱导16-18h;诱导完成后收集菌体,4℃、3,500g离心30min,除去上清液后将菌体放入-80℃冰箱保存待进行下一步的纯化。
4.目标蛋白纯化步骤如下:
(1)将收集的菌体从-80℃冰箱中取出,融化后加入30mL裂解缓冲液(His lysisbuffer)重悬,再放入-80℃冰箱中1h后取出室温融化,反复冻融两次;这样由于细胞内冰粒形成和剩余细胞液的盐浓度增高引起溶胀,从而使细胞结构破碎;
(2)将反复冻融的菌液进行超声破碎,超声条件为:开10s,关10s,工作时间1min,功率60%;重复多次使大肠杆菌完全裂解;
(3)将步骤(2)中破碎后的大肠杆菌在18,000g、4℃条件下高速离心1h,收集上清液;
(4)取出1mL对应蛋白标签的纯化介质His beads在50mL离心管中,加入5-10mLHis lysis buffer,在500-1,000g、4℃条件下低速离心1min后除去上清液。重复两次以达到平衡beads的目的;
(5)将大肠杆菌的上清液倒入平衡好的beads中,在4℃低温条件下旋转孵育1h;然后在1,500g、4℃条件下低速离心5min收集结合了目标蛋白的His beads,用5mL His lysisbuffer将His beads转移到空层析柱中,并用His wash buffer冲洗两个柱体积,使其在重力作用下自由滴落,充分清洗残留的lysis buffer和其他的细菌杂蛋白;最后依次用3mL和2mL His elute buffer进行洗脱,洗脱下来的蛋白浓缩至体积为300-400μL并进行跑胶鉴定后保存在-80℃中备用。
5.蛋白凝胶过滤层析的步骤如下:
将纯化浓缩后的蛋白在13,000g、4℃条件下高速离心10min;上样前用相应缓冲液平衡层析柱。层析柱平衡后,将样品用针管加入到进样环中,以流速0.4mL/min、压力上限1.0MPa的条件对蛋白样品进行洗脱分离,设置以每管0.5mL的体积进行收集。收集结束后,根据蛋白的UV吸收峰的结果选择需要分析的样品,并用SDS-PAGE的方法进行检测。
6.SDS-PAGE蛋白电泳及染色蛋白电泳操作步骤如下:
(1)制胶:配制不同分离浓度的SDS-PAGE胶;
(2)上样:聚丙烯酰胺凝胶配置凝聚后,安装好电泳设备。在浓缩胶的孔槽中依次加入相同体积的预染蛋白marker和蛋白样品与上样缓冲液(1×SDS loading buffer)的混合液,空槽则用上样缓冲液补齐;
(3)跑胶:可以选择恒压电泳法或恒流电泳法;以一块1.0mm厚度的胶板为例,按照90V-130V-160V的梯度进行恒压电泳,每个梯度的持续时间约为25-30min;也可以按照25mA的条件进行恒流电泳,持续时间60-100min,直到将蛋白分离至合适位置;
(4)电泳后根据需要进行考马斯亮蓝染色或转膜处理。其中,考马斯亮蓝染色操作如下:跑胶结束后,将聚丙烯酰胺凝胶从胶板上剥离并放置于考染盒中,加入适量考马斯亮蓝染液,室温孵育1h。染色结束后倒掉染液并冲洗干净,再倒入脱色液后放置在摇床上,每半小时更换一次脱色液,更换三次后脱色过夜后即可对考染后的胶进行分析。
乙酰磷酸(AcP)磷酸化实验步骤如下:
(1)AcP磷酸化体系配制所需的试剂及用量如表1所示:
表1配制AcP磷酸化体系原料表
AcP与蛋白底物的浓度比为1000:1;
(2)将配置好的AcP磷酸化体系置于37℃恒温水浴中反应1h;
(3)反应结束后,取1μL反应样品直接加入SDS Loading buffer用于后续跑胶检测,其他样品用于后续的标记实验。
磷酸化标记实验步骤如下:
1.AEP标记实验步骤如下:
经过乙酰磷酸处理得到pKdpE和pVicR后,在室温条件下,以含有8M Urea的50mMTEAB缓冲溶液作为标记反应溶剂,将40mM AEP标记试剂分别与浓度为40μM、总量约为120μg的pKdpE和pVicR孵育1h后进行酶解、除盐、LC-MS/MS检测和蛋白组学数据分析,并将未发生磷酸化的KdpE和VicR蛋白在相同的条件下同时进行标记实验和LC-MS/MS检测作为空白对照组。
2.蛋白酶解(Filter aided proteome preparation,FASP)的步骤如下:
(1)标记反应结束后,在20℃、13,000g条件下离心20min除去反应液;
(2)倒去收集管中废液后,在filter加入200μL 8M Urea,充分震荡使蛋白与尿素接触,在20℃、13,000g条件下离心30min,重复三遍;
(3)倒去收集管中废液后,在filter加入200μL 50mM DTT,充分震荡使蛋白与DTT接触,常温下反应30min;
(4)在20℃、13,000g条件下离心30min;
(5)倒去收集管中废液后,在filter加入200μL 50mM IAA,充分震荡使蛋白与IAA接触,常温避光条件下反应30min;
(6)在20℃、13,000g条件下离心30min;
(7)去掉收集管中的废液后,在filter中加入200μL 8M Urea,剧烈震荡后,于20℃、13,000g离心30min,重复三遍;
(8)去掉收集管中的废液后,在filter中加入200μL 50mM TEAB,剧烈震荡后,于20℃、13,000g离心30min,重复三遍;
(9)更换新的收集管,在filter中加入100μL 50mM TEAB溶液和含0.2μg/μLTrypsin的50mM TEAB溶液(1:50),37℃下孵育16-18h;
(10)孵育结束后,在20℃、13,000g离心20min,这时蛋白已经被酶解成肽段,可以通过filter的膜,被离心到下方的收集管中;
(11)再在filter中加入50μL 50mM TEAB溶液,适当震荡后,20℃、13,000g离心15min收集到同一个收集管中;
3.除盐步骤如下:
(1)制作Stage-tips:用C8膜堵住200μL枪头后,在里面填充5-10mg的C18粉末;
(2)活化柱子:加入200μL乙腈,200g离心1min确定合适转速和离心时间,使溶液能在3-5min离心至C18粉末上方2-5mm处,保证C18粉末全程保持湿润,重复三次;
(3)平衡柱子:加入200μL 0.1%FA,400g离心1min确定合适转速和离心时间,重复三次;
(4)上样:上一步蛋白酶解后总共得到150μL蛋白肽段样品,用甲酸将样品的pH值调节至2-4之间。将酸化后的样品加到柱子中,100g离心1min确定转速和时间,使蛋白样品与C18粉末充分结合;
(5)洗涤:加入200μL 0.1%FA,400g离心1min确定转速和时间,重复三次;
(6)洗脱:更换下层收集管后,加入100μL含0.1%FA和75%ACN的洗脱Buffer,200g离心1min确定转速和时间。再加入100μL含0.1%FA和95%ACN的洗脱Buffer,200g离心20-30min将C18柱中液体离心完全;
(7)旋干:将得到的200μL最终样品在真空离心干燥机中旋干得到蛋白肽段样品。
旋干后的样品如果不即时上机测样,需要先在-20℃冰箱内保存;待测样时取出用含2%ACN和0.1%FA的水溶液溶解后测定浓度,调整浓度后在13,000g、20℃条件下离心10min后取上清液,利用高分辨质谱仪鉴定肽段。
质谱检测与分析:
1.质谱数据收集步骤如下:
(1)将除盐后的蛋白肽段样品用20-50μL含有2%ACN和0.1%FA的溶液溶解,充分震荡混匀后,用nano-Drop测定肽段的浓度;
(2)将肽段样品调整至合适浓度后,在20℃、13,000g条件下离心10min,吸取上清液移至进样瓶中;
(3)使每个样品的进样量都在500-1,000ng之间,根据浓度确定进样体积,使进样体积在2-10μL之间。
(4)高效液相色谱分析条件如下:HPLC Easy-nLC1000(Thermo Scientific)超高压纳升液相色谱;Acclaim PepMapTM RSLC 75μm×15cm,nanoViper C18,2μm,100A(Thermo)色谱柱;流速300nL/min;A相为水溶液,B相为ACN溶液。色谱梯度:0-3min,3-7%B;3-95min,7-40%B;95-113min,40%-70%B;113-115min,70-90%B;115-125min,90%B。
(5)经色谱系统分离的蛋白质肽段样品在Orbitrap Fusion Lumos三合一高分辨质谱系统检测。质谱分析条件如下:Q-Exactive(Thermo Scientific)质谱;纳升级ESI离子源,正离子模式,喷雾电压3.5KV,毛细管温度360℃,四级杆和轨道离子阱(orbitrap)质量分析器,质量扫描范围m/z 350-1800,分辨率一级70000@m/z 200、二级17500@m/z 200,AGCtarget一级3e6、二级1e5,Maximum IT一级50ms、二级60ms,碰撞能量27%(NCE)。
2.质谱数据分析步骤如下:
将原始蛋白质组学质谱数据通过Proteome Discoverer 2.1软件进行检索与搜库分析。序列比对过程中加入目的蛋白序列库作为数据库;同时需要添加不同的蛋白固定修饰,例如半胱氨酸烷基化(+57.021Da);以及动态修饰,例如N端乙酰化(+42.011Da)、甲硫氨酸氧化(+15.995Da)和AEP(+163.0938Da)、4D-AEP(+167.1189Da)等。设定母离子质量偏差为10ppm,碎片离子偏差为0.02Da。
使用Diethyl-AEP检测AcP处理后KdpE天冬氨酸羧基磷酸化位点的实验结果如图4和表2所示。
表2KdpE与pKdpE中Asp52所在肽段二级质谱b、y离子结果对比
从图4和表2中可以看出:Diethyl-AEP标记后,检测到的pKdpE肽段覆盖率达到98.2%。对比Diethyl-AEP标记前后两组pKdpE肽段的二级质谱检测的b、y离子结果可以发现,b8 +分子量相差163.0761Da(Diethyl-AEP修饰的分子量163.0938Da),b10 +分子量相差163.0727Da,且b1 +、b5 +、b6 +和b7 +的数值都相同,由此说明该条肽段从N端第八位氨基酸也就是52位天冬氨酸开始所产生的肽段碎片离子都产生了163Da的差别,这证明Diethyl-AEP标记试剂能成功鉴定出KdpE中52位天冬氨酸的羧基磷酸化。
实施例3、使用Diethyl-AEP分析大肠杆菌蛋白组中AcP处理后VicR的天冬氨酸羧基磷酸化位点
采用与实例2同样的标记反应条件和质谱检测方法对标记反应后的pVicR进行检测。使用Diethyl-AEP检测AcP处理后VicR天冬氨酸羧基磷酸化位点的实验结果如图5和表3所示。
表3 VicR和pVicR中Asp52所在肽段二级质谱b、y离子结果对比
从图5和表3中可以看出:Diethyl-AEP标记后,检测到的pVicR肽段覆盖率达到89.4%。对比Diethyl-AEP标记前后两组pVicR肽段的二级质谱检测的b、y离子结果可以发现,b2 +、b4 +、b7 +、b9 +以及y6 +、y10 +、y11 +、y12 +的数值都相同。说明该条肽段从N段至第九位氨基酸和从C端至第十二位氨基酸所产生的的肽段碎片离子都相同,因此修饰位点位于N端第十位至第十三位氨基酸之间,根据反应调节蛋白VicR的磷酸化特性以及Diethyl-AEP标记反应的特点,推测修饰位点为第十二位氨基酸也就是Asp52,证明Diethyl-AEP标记试剂能成功鉴定出VicR中52位天冬氨酸的羧基磷酸化。
实施例4、使用Diethyl-AEP分析大肠杆菌蛋白组中体外激酶反应处理后VicR/VicK双组分系统的天冬氨酸羧基磷酸化位点
以VicRK系统为例模拟双组分系统,通过原核表达所得反应调节蛋白VicR与组氨酸激酶VicK的体外激酶反应得到pVicR后,设置未加ATP而其他反应条件相同的VicRK体系作为空白对照组,以与实例2相同条件进行标记实验和质谱检测。使用Diethyl-AEP检测体外激酶反应处理后VicR/VicK双组分系统的天冬氨酸羧基磷酸化位点的实验结果如图6和表4所示。
表4 VicR与pVicR中Asp192所在肽段二级质谱b、y离子结果对比
从图6和表4中可以看出:Diethyl-AEP标记后,检测到的pVicR肽段覆盖率达到68.1%,所识别的修饰位点除了Asp52外,还发现了一个新的修饰位点Asp192,证明Diethyl-AEP标记试剂能成功鉴定出体外激酶反应中VicR天冬氨酸的羧基磷酸化。
Claims (10)
2.如权利要求1所述的含稳定同位素的蛋白质羧基磷酸化标记试剂的制备方法,其特征在于包括以下步骤:
(1)叔丁氧羰基保护的溴胺的制备:低温下将二碳酸二叔丁酯、溴胺化合物置于有机溶剂中,逐滴加入有机碱后将反应混合物升温到室温继续搅拌;然后除去溶剂,将所得剩余物纯化后即可制得叔丁氧羰基保护的溴铵;
(2)叔丁氧羰基保护的含稳定同位素的氨基膦酸酯的制备:将钠氢悬浮在无水有机溶剂中,然后逐滴加入含稳定同位素的亚磷酸酯,将所得混合物在室温下反应后加热回流,接下来在低温下加入步骤(1)中制备的叔丁氧羰基保护的溴铵,将所得混合物在室温下继续反应,最后用水淬灭反应,将淬灭后所得产物纯化后即可制得叔丁氧羰基保护的含稳定同位素的氨基膦酸酯;
(3)含稳定同位素的氨基膦酸酯的制备:将步骤(2)制备的叔丁氧羰基保护的含稳定同位素氨基膦酸酯溶于有机溶剂后加入酸,所得反应混合物在室温下搅拌反应,除去溶剂后将所得反应剩余物纯化后即可制得含稳定同位素的氨基膦酸酯。
3.如权利要求2所述的含稳定同位素的蛋白质羧基磷酸化标记试剂的制备方法,其特征在于:步骤(1)和步骤(3)中所述的有机溶剂选自乙酸乙酯,氯仿或二氯甲烷中的一种。
4.如权利要求2所述的含稳定同位素的蛋白质羧基磷酸化标记试剂的制备方法,其特征在于:步骤(1)中所述的溴胺化合物为溴甲胺,2-溴乙胺,3-溴丙胺,4-溴丁胺,5-溴戊胺或6-溴己胺中的一种;所述的有机碱为吡啶、二乙胺,二异丙胺或三乙胺中的一种;所述的室温搅拌反应的时间为16-24小时。
5.如权利要求2所述的含稳定同位素的蛋白质羧基磷酸化标记试剂的制备方法,其特征在于:步骤(2)中所述的无水有机溶剂为无水N,N-二甲基甲酰胺,无水1,4-二氧六环或无水四氢呋喃中的一种;所述的含稳定同位素的亚磷酸酯为亚磷酸二甲酯,亚磷酸二乙酯,亚磷酸二丙酯,亚磷酸二丁酯或亚磷酸二戊酯中的一种。
6.如权利要求2所述的含稳定同位素的蛋白质羧基磷酸化标记试剂的制备方法,其特征在于:步骤(3)中所述的酸为盐酸,甲酸,醋酸或三氟乙酸中的一种。
7.如权利要求1所述的含稳定同位素的蛋白质羧基磷酸化标记试剂在鉴定蛋白质羧基磷酸化修饰中的应用,其特征在于具体步骤如下:
(1)蛋白乙酰磷酸化反应:将蛋白质加入到乙酰磷酸化试剂中进行恒温反应;
(2)标记反应:将步骤(1)中经过乙酰磷酸处理过的蛋白质与缓冲溶液和标记试剂一起进行标记反应获得标记蛋白产物;其中,所述缓冲溶液为碱性缓冲溶液;标记反应温度为0℃至室温;标记反应的时间为0.5h-1h;
(3)LC-MS/MS检测:将步骤(2)中获得的标记蛋白产物除盐后进行LC-MS/MS检测,记录LC-MS/MS谱图;
色谱条件如下:采用超高压纳升液相色谱;流速为300nL/min;A相为水溶液,B相为ACN溶液;
质谱参数如下:采用纳升级ESI离子源,正离子模式,喷雾电压为3.5KV,毛细管温度为360℃;采用飞行时间或轨道离子阱(orbitrap)质量分析器,质量扫描范围m/z 350-1800;
(4)蛋白组学数据分析:将步骤(3)中获得的LC-MS/MS谱图进行检索与序列比对分析;其中,在序列比对过程中需加入目的蛋白序列库作为数据库,同时还需要添加不同的蛋白固定修饰和动态修饰。
8.如权利要求7所述的含稳定同位素的蛋白质羧基磷酸化标记试剂在鉴定蛋白质羧基磷酸化修饰中的应用,其特征在于:步骤(1)中所述的恒温反应温度为37℃,所述的恒温反应时间为1h。
9.如权利要求7所述的含稳定同位素的蛋白质羧基磷酸化标记试剂在鉴定蛋白质羧基磷酸化修饰中的应用,其特征在于:步骤(2)中所述的碱性缓冲溶液为四乙基溴化铵溶液。
10.如权利要求7所述的含稳定同位素的蛋白质羧基磷酸化标记试剂在鉴定蛋白质羧基磷酸化修饰中的应用,其特征在于:步骤(2)中所述的标记反应中标记试剂和蛋白的浓度比例为10-1000:1。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101137724A (zh) * | 2005-03-08 | 2008-03-05 | 西巴特殊化学制品控股公司 | 涂覆有特定膦酸酯的金属氧化物纳米粒子 |
CN106596967A (zh) * | 2016-11-25 | 2017-04-26 | 厦门大学 | 一种稳定同位素磷酰化标记蛋白质的定量方法 |
CN106608890A (zh) * | 2015-10-21 | 2017-05-03 | 厦门大学 | 一类磷酸化精氨酸类似物及其合成方法与应用 |
CN106749402A (zh) * | 2016-11-25 | 2017-05-31 | 厦门大学 | 一种稳定同位素标记手性磷试剂及其制备方法与应用 |
CN107722166A (zh) * | 2017-09-22 | 2018-02-23 | 陈晨特 | 一种磷氮协同自阻燃丙烯酸乳液及其制备方法 |
JP2021080183A (ja) * | 2019-11-14 | 2021-05-27 | 田辺三菱製薬株式会社 | 新規リゾホスファチジン酸誘導体 |
CN113999125A (zh) * | 2021-11-09 | 2022-02-01 | 苏州昊帆生物股份有限公司 | 2-甲基乙胺的制备方法 |
-
2022
- 2022-04-14 CN CN202210386769.6A patent/CN114736238B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101137724A (zh) * | 2005-03-08 | 2008-03-05 | 西巴特殊化学制品控股公司 | 涂覆有特定膦酸酯的金属氧化物纳米粒子 |
CN106608890A (zh) * | 2015-10-21 | 2017-05-03 | 厦门大学 | 一类磷酸化精氨酸类似物及其合成方法与应用 |
CN106596967A (zh) * | 2016-11-25 | 2017-04-26 | 厦门大学 | 一种稳定同位素磷酰化标记蛋白质的定量方法 |
CN106749402A (zh) * | 2016-11-25 | 2017-05-31 | 厦门大学 | 一种稳定同位素标记手性磷试剂及其制备方法与应用 |
CN107722166A (zh) * | 2017-09-22 | 2018-02-23 | 陈晨特 | 一种磷氮协同自阻燃丙烯酸乳液及其制备方法 |
JP2021080183A (ja) * | 2019-11-14 | 2021-05-27 | 田辺三菱製薬株式会社 | 新規リゾホスファチジン酸誘導体 |
CN113999125A (zh) * | 2021-11-09 | 2022-02-01 | 苏州昊帆生物股份有限公司 | 2-甲基乙胺的制备方法 |
Non-Patent Citations (1)
Title |
---|
DAGMAR C. KAPELLER ET AL: "Preparation of Enantiopure Chiral Amino-[D1]methyllithium Compounds and Determination of Their Micro- and Macroscopic Configurational Stabilities", CHEM. EUR. J., vol. 15, pages 5729 - 5739, XP071829597, DOI: 10.1002/chem.200802668 * |
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