CN114031540A - 一种定量检测生物硫醇的氟标签及其用途 - Google Patents
一种定量检测生物硫醇的氟标签及其用途 Download PDFInfo
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Abstract
本发明提供一种定量检测生物硫醇的氟标签及其用途,利用该标签进行生物硫醇的区分与定量,并进一步对细胞内生物硫醇进行检测。该活性氟标签与生物硫醇反应生成稳定的硫酯产物,其具有可区分的19F化学位移。因此,通过1D 19F‑NMR可以同时区分并定量这些生物硫醇。利用该氟标签进一步研究了活细胞内/外谷胱甘肽的变化,并观测到正常哺乳动物细胞和肿瘤细胞之间GSH稳定性的显著差异。值得注意的是,GSH能在肿瘤细胞外膜及其裂解物中有效水解。相反,GSH在正常细胞中保持稳定。因此我们进一步研究了肿瘤细胞裂解物中GSH的水解产物。所提出的方法具有良好的可重复性以及宽的线性范围,不受其他背景因素的干扰,操作简便,具有广阔的应用前景。
Description
技术领域
本发明涉及复杂体系中生物样品分析,具体涉及利用氟标签并通过核磁共振氟谱进行生物硫醇的定量分析以及细胞内GSH代谢追踪。
背景技术
生物硫醇如谷胱甘肽(GSH)、半胱氨酸(Cys)、同型半胱氨酸(Hcy)和硫化氢(H2S)在细胞代谢和维持氧化还原稳态等多种生理和病理过程中发挥重要作用。半胱氨酸不仅是蛋白质合成的重要氨基酸,也是谷胱甘肽合成的前体化合物。半胱氨酸的含量变化可能导致一些疾病,包括生长缓慢、肝损伤、阿兹海默症、帕金森等疾病相关。同型半胱氨酸与阿兹海默症、骨质疏松等疾病相关。谷胱甘肽是细胞内重要的还原剂,其也与多种疾病相关。谷胱甘肽含量缺乏可能与HIV,心脏病、中风、癌症、慢性呼吸道疾病以及糖尿病等疾病相关。硫化氢的水平异常也与亨廷顿病、帕金森病以及阿兹海默症等疾病相关(Chem.Soc.Rev.,2015,44:4596~4618)。由于这些生物硫醇的含量与多种疾病相关,因此H2S、Cys、Hcy和GSH的鉴别和定量是生物分子分析方法发展的主要方向之一。近年来,生物硫醇的分析方法主要包括荧光光谱法(Angew.Chem.Int.Ed.2017,56:13188~13198)、分光光度法(Angew.Chem.Int.Ed.2011,50:6283~6286)、质谱法(Anal.Chem.2010,82:6926~6932)、高效液相色谱法(J.Chromatogr.B 2011,879,1290~1307)和毛细管电泳法(Anal.Chem.2002,74:1349~1354.)等,其中,荧光光谱法最常用。目前,核磁共振波谱作为代谢组学研究中的重要技术已经显示出生物硫醇分析的优势。19F的天然丰度为100%(Chem.Rev.2015,115:1106-1129.)并且具有与1H类似的灵敏度(约为1H的83.4%),并且19F-NMR的化学位移范围宽,不易发生信号重叠,因此19F-NMR在生物体系中具有广泛应用。
发明内容
本发明旨在通过19F-NMR利用活性19F标签在生理条件下区分并定量GSH、Hcy、Cys和H2S,并应用于细胞内生物硫醇检测。在此基础上,我们进一步研究了细胞层次上GSH的代谢。
本发明提供了一种定量检测生物硫醇的氟标签,具体结构式如下,
其结构中包含三氟甲基(CF3)以及巯基反应基团—N-羟基琥珀酰亚胺酯(NHS)基团。通过氟标签与生物硫醇中巯基发生反应从而生成具有氟信号的反应产物,利用不同分子产生氟信号的化学位移的不同从而达到区分的目的。
本发明首先将不同浓度的生物硫醇标准液与氟标签混合,进行不同生物硫醇与氟标签反应产物化学位移对比,并进行浓度-峰强度拟合;将待测样品与氟标签混合,利用19F-NMR进行检测,依据所得浓度-峰强度线性拟合结果得出待测样品中各生物硫醇中的含量。
进一步,本发明利用氟标签进行了生物硫醇混合物体系中生物硫醇的区分,如下:
将氟标签溶于DMF溶液中,配制为浓度为100mM的母液,取5μL氟标签加入到分别含有0.2mM的GSH、Cys、Hcy以及Na2S的磷酸盐缓冲液(50mM,pH 7.5)中,得到含有1mM氟标签的检测体系;反应2h后,采集19F-NMR。接着取5μL氟标签加入浓度均为0.2mM的GSH、Cys、Hcy以及Na2S的混合体系中;反应2h后,采集19F-NMR。
进一步地,上述所述样品GSH、Cys、Hcy以及Na2S均配制于超纯水中,母液浓度为100mM。
本发明提供了一种复杂体系如混合物中生物硫醇的定量检测方法,如下:
首先将19F标签与含有不同浓度生物硫醇的标准样品分别进行反应,得到浓度与产物峰强度的标准曲线。在含有不同浓度的四种生物硫醇的混合体系中,向其中加入5μL氟标签,得到含有1mM氟标签的检测体系,于25℃反应4h后,反应完全,采集19F-NMR。样品测试多组,每组重复3次。根据标准曲线以及测得的混合样品中的产物峰强度可以分别计算出混合样品中实际的样品浓度。
进一步地,上述定量研究中,所用核磁参数为:采集次数(ns)为128次,采样温度为25℃,数据点数(TD)为16k,弛豫延迟时间(D1)为2S。
进一步地,上述所选生物硫醇浓度为0.05-0.50mM。
进一步地,上述步骤中所用方法进一步检测了19F标签的检测限度可低至2μM。
本发明提供了一种检测活细胞内外GSH的方法,如下:
HEK293T、HeLa细胞贴壁生长培养,置于含有10mL DMEM培养基(10%胎牛血清+1%青-链霉素)的10cm培养皿,于37℃,5%CO2中培养。其中HEK293T细胞为正常细胞,HeLa细胞为肿瘤细胞。当细胞覆盖90%时,移除培养基,并用PBS洗两次,胰酶消化,将细胞收集,计数。将收集的细胞重悬于PBS中,向其中加入一定量氟标签至终浓度为1mM,采集不同时间的19F-NMR谱图。
进一步地,上述重悬细胞所用PBS中补加了0.5%的葡萄糖。
本发明提供了一种细胞裂解物中GSH水解途径的检测方法,如下:
按上述方法进行HeLa细胞培养并收集,计数。将收集的细胞用裂解缓冲液裂解细胞,裂解完成后,得到细胞裂解物,向细胞裂解物中加入终浓度为0.1mM的19F标签,采集不同时间的19F-NMR谱图。
进一步地,上述所用细胞裂解液为商品化细胞裂解液,其配方为20mM Tris-HCl(pH 7.4),150mM氯化钠,1%NP-40,5%甘油,2.5mM焦磷酸钠,1mMβ-甘油磷酸钠,1mM乙二胺四乙酸。
本发明具有如下优点以及效果:
本发明构建了一种有效的核磁分析方法,该方法通过19F标签与生物硫醇之间的共价作用来区分并定量不同生物硫醇分子。该方法具有较低检测限,良好的可重复性以及无背景干扰。该方法进一步可应用于检测细胞内生物硫醇,并利用该方法我们观测到了细胞裂解液中GSH的降解,具有广泛的应用前景。
附图说明
图1(a)为本发明中氟标签与生物硫醇的反应机制示意图,(b)为本发明中氟标签与生物硫醇反应产物分析。
图2为本发明中氟标签与不同生物硫醇分子反应后的19F-NMR谱图;
图3为本发明中根据浓度与信号强度绘制得到的标准曲线;
图4为本发明中混合体系中浓度定量结果;
图5为本发明中19F标签检测生物硫醇混合体系中检测限度;
图6为本发明中19F标签检测细胞样品中生物硫醇的氟谱图;
图7为本发明中19F标签对细胞存活率的测试;
图8为本发明中19F标签检测HeLa细胞裂解物中GSH随时间的变化图。
具体实施方式
以下结合附图实施例对本发明进行进一步阐释。
实施例1,氟标签的合成:
将2-(三氟甲基)苯甲酸(3.8g,20mmol)、N-羟基琥珀酰亚胺(2.3g,20mmol)、N,N'-二环己基二亚胺(4.5g,22mmol)混合在40mL二氯甲烷中。得到的溶液在室温下搅拌4h,然后过滤。蒸发滤液,用正己烷和乙醚洗涤,得到目标化合物2-三氟甲基苯甲酸琥珀亚胺酯为白色固体(5.0g,产率82%左右)。1H NMR(400MHz,CDCl3):δ8.13(d,J=7.4Hz,1H),7.87(d,J=7.5Hz,1H),7.79-7.69(m,2H),2.92(s,4H)。13C NMR(101MHz,CDCl3):δ168.83,161.07,133.34,132.01,131.46,130.16(q),127.27(q),125.14,122.72(q),25.67。
实施例2,氟标签与不同生物硫醇的反应表征:
将氟标签(100mM)溶于DMF溶液中,取5μL氟标签加入到分别含有0.2mM的GSH、Cys、Hcy以及Na2S的磷酸盐缓冲液(50mM,pH 7.5)中,得到含有1mM氟标签的检测体系,氟标签与生物硫醇的反应机制,见附图1;同样取5μL氟标签加入浓度均为0.2mM的GSH、Cys、Hcy以及Na2S的混合体系中;反应2h后,采集19F-NMR谱图,与氟标签与单个生物硫醇反应谱图对比,见附图2。
实施例3,混合体系中生物硫醇的同时定量研究:
1)使用实施例2中的方法得到氟标签与不同浓度生物硫醇的峰强度与浓度之间关系的标准曲线,见附图3。其中测试生物硫醇的浓度为0.05mM-0.50mM。
2)配制三组含有不同浓度的生物硫醇的待测样品,样品1:0.2mM GSH,0.15mMNa2S,0.05mM Hcy,0.15mM Cys;样品2:0.05mM GSH,0.10mM Na2S,0.15mM Hcy,0.20mM Cys;样品3:0.10mM GSH,0.10mM Na2S,0.10mM Hcy,0.10mM Cys,加入5μL氟标签,反应4h,反应完全,每组样品重复三次。根据所测的待测样品的产物峰强度与标准曲线,反算出测得浓度并与加入的实际浓度进行对比,结果见附图4,结果表明利用该方法测得的生物硫醇各组分的含量与实际浓度具有高度一致性,该方法具有较好的准确性。
3)配制终浓度为2μM的GSH、Cys、Hcy、Na2S的四种生物硫醇混合物与19F标签反应,采集氟谱,结果见附图5,结果表明该方法能够对浓度低至2μM的样品进行检测分析。
实施例4,氟标签在不同细胞内生物硫醇的检测结果:
HEK293T,HeLa细胞贴壁生长培养,置于含有10mL DMEM培养基(10%胎牛血清+1%青-链霉素)的10cm培养皿,于37℃,5%CO2中培养。细胞覆盖90%时,移除培养基,并用PBS洗两次,胰酶消化,将细胞收集,计数。将收集的细胞重悬于PBS(含有0.5%葡萄糖)中,向其中加入一定量氟标签至终浓度为1mM,采集不同时间的19F-NMR谱图,见附图6。正常细胞(HEK293T)中只有GSH与19F标签的产物生成,而肿瘤细胞(HeLa)中除GSH与19F标签反应产物生成外,还有Cys-Gly二肽与19F标签生成产物以及Cys与19F标签生成产物。
实施例5,氟标签对细胞的毒性测试:
按照实施例4中步骤进行HeLa细胞培养与收集,并将细胞重悬于PBS(含有0.5%葡萄糖)中,向其中加入终浓度为1mM的19F标签,将样品置于室温6h后,进行台盼蓝(0.4%)染色,染色结果如附图7所示,细胞存活率在95%以上,说明本发明中19F标签对细胞是较为安全的。
所述染色比例为细胞:台盼蓝为9:1,染色时间为3min。
实施例6,细胞裂解物内GSH的代谢路径研究:
按照实施例4中进行HeLa细胞培养并收集,计数。将收集的细胞用裂解缓冲液裂解细胞,于0℃裂解15分钟,得到细胞裂解物,向细胞裂解物中加入终浓度为1mM的19F标签,采集不同时间的19F-NMR谱图,结果见附图8。结果表明利用本发明中19F标签可以观测到裂解物中GSH的水解途径。
Claims (8)
2.一种权利要求1所述的氟标签的用途,其特征在于:用于区分并定量生物硫醇混合物样品中生物硫醇各组分含量,包括如下步骤:
(1)称取氟标签2-三氟甲基苯甲酸琥珀亚胺酯加入到DMF中配制浓度为100mM的溶液;
(2)将不同浓度的生物硫醇标准液与氟标签混合,进行不同生物硫醇与氟标签反应产物化学位移对比,并进行浓度-峰强度拟合;
(3)将待测样品与氟标签混合,利用19F-NMR进行检测,依据步骤(2)中所得浓度-峰强度线性拟合结果得出待测样品中各生物硫醇中的含量。
3.根据权利要求2所述的氟标签的用途,其特征在于:
所述生物硫醇为谷胱甘肽、L-半胱氨酸、同型半胱氨酸、硫化氢;
所述生物硫醇标准溶液的浓度为50~500μM,待测样品为以上四种生物硫醇不同浓度的混合物,标准溶液以及待测样品溶液均为超纯水配制为浓度为100mM的母液;
所述反应体系均于pH值为7.5的50mM磷酸缓冲液中进行。
4.根据权利要求2所述的氟标签的用途,其特征在于:用于检测细胞内生物硫醇,包括如下步骤:
HEK293T、HeLa细胞贴壁生长培养,置于含有10mL DMEM培养基的10cm培养皿,于37℃,5%CO2中培养;细胞覆盖90%时,移除培养基,并用PBS洗两次,胰酶消化,将细胞收集,计数;将收集的细胞重悬于PBS(含有0.5%葡萄糖)中,向其中加入一定量氟标签至终浓度为1mM,采集不同时间的19F-NMR谱图。
5.根据权利要求4所述的氟标签的用途,其特征在于:DMEM培养基中包含10%胎牛血清+1%青-链霉素。
6.根据权利要求4所述的氟标签的用途,其特征在于:PBS中含有0.5%葡萄糖。
7.根据权利要求2所述的氟标签的用途,其特征在于:用于检测细胞裂解物中谷胱甘肽水解产物追踪,包括如下步骤:
按照权利要求4中细胞培养步骤进行细胞培养,收集并计数,将收集的细胞用细胞裂解缓冲液于0℃裂解15min,向其中加入终浓度为1mM的氟标签,采集不同时间的19F-NMR谱图,观测到细胞裂解物中谷胱甘肽的水解产物。
8.根据权利要求7所述的氟标签的用途,其特征在于:所述细胞裂解缓冲液配方为pH值为7.4的20mM Tris-HCl、150mM氯化钠、1%NP-40、5%甘油、2.5mM焦磷酸钠、1mMβ-甘油磷酸钠、1mM乙二胺四乙酸。
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