CN109187407B - 一种5-(4-甲氧基苯基)-1-苯基-1h-三唑与血清作用的差异蛋白检测方法 - Google Patents
一种5-(4-甲氧基苯基)-1-苯基-1h-三唑与血清作用的差异蛋白检测方法 Download PDFInfo
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Abstract
本发明涉及一种5‑(4‑甲氧基苯基)‑1‑苯基‑1H‑三唑与血清作用的差异蛋白检测方法,包括:将血清样品与5‑(4‑甲氧基苯基)‑1‑苯基‑1H‑1,2,3‑三唑化合物混合;除去高丰度蛋白;Bradford法定量测定蛋白浓度;进行双向电泳实验;胶片图像处理及质谱鉴定等步骤。本发明还提供一种5‑(4‑甲氧基苯基)‑1‑苯基‑1H‑1,2,3‑三唑的制备方法。
Description
技术领域
本发明属于有机合成及生物化学领域,具体涉及一种5-(4-甲氧基苯基)-1-苯基-1H-三唑与血清作用的差异蛋白检测方法。
背景技术
1,2,3-三氮唑化合物以其独特的五元芳香含氮杂环结构,可与其他取代基相缀合或并合形成多种类型具有药物活性的衍生物,又表现出低毒性及良好的生物活性,已在材料化学、药物化学、有机化学和有机金属化学等许多领域有广泛的应用,并愈来愈受到研究者的青睐。而蛋白质组学是一门在蛋白质水平上认识生命机理的学科,其学术理念和相关技术方法已被广泛应用于生命科学的各个领域,涉及多种重要的生物学现象,并已成为人类重大疾病诊断、治疗和寻找药物靶点的有效方法之一。小分子药物的作用靶点通常是蛋白质,而蛋白质组学是一种应用化学小分子来检测整个目标蛋白质组的手段,从整体水平上,观察蛋白质组的变化,发现差异表达蛋白并研究它的的生物功能以及与小分子的作用机理,从而找到新的药物靶点或者发现具有药用价值的小分子。
人体血液中的蛋白质几乎与机体中所有的细胞、组织、器官有关,血液蛋白的动态变化可以直接反应机体的健康情况,是研究各类疾病相关标志物最有价值的标本。本发明化合物5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑通过在模拟人生理温度条件下,进行血清与化合物相互结合的体外培养,并采用蛋白质组学研究方法结合MALDI-TOF-MS技术开展在化合物存在时的血清蛋白质组学研究,分析差异表达蛋白,寻找特定疾病的特异血清蛋白标志物或者发现新的疾病相关蛋白质,为疾病的诊断或治疗提供新的依据,为5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑可能作为新药物的开发提供更加直观的理论基础。
发明内容
本发明提供一种5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑与血清作用检测差异蛋白的方法,其特征在于包括如下步骤:
(1)将血清蛋白与5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物溶液混合均匀得样品a,恒温培养得样品b;
(2)去除样品b中的高丰度蛋白得样品c
(3)用Bradford法对样品c的蛋白浓度进行定量。
(4)进行双向电泳实验:
①按每胶条蛋白质上样量1.3mg,上样总体积为455uL的标准水化上样,进行第一向等电聚焦;
②第一向等电聚焦完毕后,将胶条平衡后转到聚丙烯酰胺凝胶进行第二垂直电泳实验;
③电泳结束后进行考马斯亮蓝染色,脱色,保存,扫描分析;
(5)胶片图像处理及质谱鉴定
①凝胶图像分析,筛选出表达异常的蛋白点并做上标记;
②差异蛋白点的酶解及质谱鉴定;
(6)结果
利用ImageMaster5.0凝胶图像分析软件获得21个差异表达蛋白质点,再通过NCBInr的Blast的检索,确定了其中1-6、8-10、12-13、15-18、21号差异表达蛋白的信息(见下表)。
所述步骤(1)中5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物溶液为5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物的甲醇溶液,样品a中5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物的浓度为2.0×10-3mol/L;恒温培养:温度为37℃,转速为r=280rpm,时间为1h;所述血清蛋白优选人血清蛋白;
所述步骤(2)中去除高丰度蛋白的方法优选按照AndyBio试剂盒要求去除高丰度蛋白;
所述步骤(4)中胶条规格为24cm非线性IPG胶条IPG pH=3~10NL。
本发明的另一实施方案提供5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑在与血清蛋白作用确定差异蛋白中的应用。
本发明的另一实施方案提供一种制备5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑的方法,其特征在于包括如下步骤:
室温下,将叠氮苯与对甲氧基苯乙烯溶于有机溶剂中,加入SmI2,搅拌反应2.5小时后得到5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑;
所述有机溶剂为THF与HMPA体积比为1:1的混合溶液;叠氮苯、对甲氧基苯乙烯、SmI2的摩尔比为1:1.1-1.3:2.0。
与现有技术相比,本发明的优点在于:(1)本发明建立了5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物与人血清作用的差异蛋白检测的双向电泳研究方法,优点是样品处理时间短,操作简单,获得的图谱清晰,重复性好;共获得21个差异表达蛋白,并成功鉴定了16个,对进一步了解5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑作为活性化合物小分子参与转运和代谢过程中相关的血液蛋白信息及相关的代谢网络,及更为深入研究开发及利用5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑奠定科学合理的理论基础;(2)本发明提供了一种以SmI2催化叠氮苯与对甲氧基苯乙烯制备5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑的全新方法,该方法操作简便,收率高。
附图说明
图1为5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑的结构图;
图2为人血清样品双向电泳图谱;
图3为人血清样品与5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物相互作用的双向电泳图谱;
图4为1号蛋白一级质谱图;
图5为1号蛋白肽段分子量为1994.1335(+1)的二级图谱;
图6为2号蛋白一级质谱图;
图7为2号蛋白肽段分子量为1478.7638(+1)的二级图谱;
图8为3号蛋白一级质谱图;
图9为3号蛋白肽段分子量为2254.1389(+1)的二级图谱;
图10为4号蛋白一级质谱图;
图11为4号蛋白肽段分子量为1773.8827(+1)的二级图谱;
图12为5号蛋白一级质谱图;
图13为5号蛋白肽段分子量为1646.9363(+1)的二级图谱;
图14为6号蛋白一级质谱图;
图15为6号蛋白肽段分子量为1874.9916(+1)的二级图谱;
图16为8号蛋白一级质谱图;
图17为8号蛋白肽段分子量为980.54681的二级图谱;
图18为9号蛋白一级质谱图;
图19为9号蛋白肽段分子量为1884.0687(+1)的二级图谱;
图20为12号蛋白一级质谱图;
图21为12号蛋白肽段分子量为1874.9916(+1)的二级图谱;
图22为13号蛋白一级质谱图;
图23为13号蛋白肽段分子量为2451.4080(+1)的二级图谱;
图24为15号蛋白一级质谱图;
图25为15号蛋白肽段分子量为1179.6797(+1)的二级图谱;
图26为16号蛋白一级质谱图;
图27为16号蛋白肽段分子量为1716.9952的二级图谱;
图28为17号蛋白一级质谱图;
图29为17号蛋白肽段分子量为1529.8212(+1)的二级图谱;
图30为18号蛋白一级质谱图;
图31为18号蛋白肽段分子量为1623.8837(+1)的二级图谱;
图32为21号蛋白一级质谱图;
图33为21号蛋白肽段分子量为2771.3528(+1)的二级图谱。
具体实施方式
为了便于对本发明的进一步理解,下面提供的实施例对其做了更详细的说明。但是这些实施例仅供更好的理解发明而并非用来限定本发明的范围或实施原则,本发明的实施方式不限于以下内容。
实施例一、血液样品材料
选用健康人血液标本男女各100例,平均年龄17±1岁。所有血样标本均在清晨空腹状态下采集,抽取体检者静脉血置于干净无菌的试管中,静置2小时后快速离心分层,保存于-80℃超低温冰箱。
实施例二、血清样品与1,2,3-三氮唑化合物混合培养
随机抽取12例血液样本(男女各6例)溶解收集上层血清蛋白,加入5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物储备液(储备液为5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑的甲醇溶液),使其在血清样品中的浓度为2.0×10-3mol/L,h混合均匀。取2ml混合血清样品和2ml对照血清样品放置37℃恒温箱,在转速280rpm条件下,孵育1小时。
实施例三、去除血清中高丰度蛋白
孵育后的血清样本均按AndyBio试剂盒要求进行操作。具体如下:
1)取100ul冰浴的试剂A加入到20ul血清样本中混合均匀。
2)放入-20℃冰箱冷藏90min。
3)取出样品并在转速为15000g,4℃的条件下离心20min。
4)小心去除上清液。
5)取1ml冰浴的试剂B到沉淀中,混合均匀。
6)混合液置于冰水中15min。
7)然后在转速15000g,4℃的条件下离心混合液20min。
8)小心去除上清液。
9)沉淀样品自然风干。
10)按10mg/mL的比例加入蛋白裂解液使风干样品完全溶解,所得白色溶液即为去除高丰度蛋白样品。
实施例四、Bradford法测样品溶液中的蛋白浓度
1)配制Bradford溶液。
2)配制标准蛋白溶液,精确称量牛血清蛋白样品用超纯水配制成1mg/mL溶液。
3)用Bradford溶液将标准蛋白储备液稀释成0、2、4、6、8、10μg/mL的系列浓度。
4)利用紫外分光光度法测定系列标准蛋白溶液在595nm处的吸光度值,并根据标准蛋白浓度和相应的吸光度值做成标准曲线方程。
5)测定目标蛋白样品溶液在595nm条件下的吸光度值,制作标准曲线,计算出样品中蛋白浓度。
实施例五、双向电泳实验步骤
1)水化上样
在室温20℃下,按每胶条蛋白质上样量1.3mg,上样总体积为455uL的标准在一次性水化盘内采用胶内水化上样,时间为18h,胶条规格为24cm非线性IPG胶条IPG pH=3~10NL。
2)第一向等电聚焦
在20℃条件下,将水化结束后的胶条迅速转移至Ettan IPGphor3聚焦仪上。按表1中的参数运行:
表1等电聚焦参数
步骤 | 项目 | 电压(v) | 时间(h) |
1 | 除盐 | 250 | 4 |
2 | 除盐 | 500 | 2 |
3 | 除盐 | 1000 | 1 |
4 | 升压 | 1000-8000 | 3 |
5 | 正式聚焦 | 8000 | 11万vlu |
6 | 保护 | 1000 | 任意 |
3)制胶
配制12.5%的垂直板聚丙稀酰胺凝胶,将胶母液注入到干净无水的玻璃夹层中,上部留1cm左右的空间,用饱和的正丁醇封面。
4)胶条平衡
等电聚焦结束后,将IPG胶条先后在平衡液Ⅰ(每10ml平衡缓冲液现加入0.125g二巯基苏糖醇)和平衡液Ⅱ(每10ml平衡缓冲液现加入0.125g碘乙酰胺)中震荡15min。所述平衡液I和II的基本配方均为:PH=8.8,1.5M tris-HCl,尿素6M,甘油30%,SDS 2%,溴酚蓝0.002%。
5)第二向-十二烷基硫酸钠聚丙稀酰胺凝胶电泳(SDS-PAGE)
平衡后的胶条转至聚丙稀酰胺凝胶上方,排除胶条下所残留的气泡,用琼脂糖封胶液将胶条稳住。待琼脂糖凝固后,将凝胶玻璃板固定于垂直电泳模具上,插入电泳槽。循环水浴设置为16℃,按电功率5W运行1h,电功率7W运行至溴酚蓝指示剂到达凝胶底端约5mm处。
6)染色及脱色
将凝胶片切角作标记,再放入考马斯亮兰染色液中,水平震荡10h左右。染色后的凝胶片放入脱色液中,水平震荡20min,重复两次。再用超纯水洗脱至凝胶片背景色消失。
实施例六、图像处理及质谱鉴定
1)凝胶图像分析
采用Image Scanner III扫描仪扫描凝胶图谱,扫描后的图像用Image Master5.0分析软件进行分析,对比筛选出表达异常的蛋白点并做上标记。
2)制作干胶粒
挖出与标记蛋白相匹配的蛋白点,放入合适的EP管中并做好标记。依次加入超纯水、脱色液和乙腈进行洗涤脱色,得到的胶粒置于超净台小风吹干。
3)蛋白样品酶解
根据干胶粒体积,加入5-6ul的胰蛋白酶溶液(20ng/uL)至完全覆盖住胶粒,置于4℃冰箱1h,使胶粒充分吸收酶液。去除多余的酶液,再根据胶粒大小补加5-7ul的胰蛋白酶buffer,于37℃酶解13h左右。酶解后的蛋白样品在10000g,常温下离心5min,收集酶解液用于质谱鉴定。
4)目标蛋白的质谱鉴定及数据库检索
采用基质辅助激光解析飞行时间质谱进行差异蛋白的鉴定,测试前需以基质峰、酶自切峰进行校正。仪器参数设置:Nd:YAG激光器,335nm,200Hz激光激发。将原始质谱数据输入MatrixScience网站(http://www.matrixscience.com)进行搜索,可得到理论上与酶解肽段相匹配的蛋白。主要搜索参数:数据库(Database):NCBIprot;酶(Enzyme):胰蛋白酶(Trypsin);种类(Taxonomy):人(Homo sapiens);固定修饰(Fixed modification):脲甲基半胱氨酸Carbamidomethyl(C);可变修饰(Variablemodification):氧化(Oxidation)。
图2是对照组人血清样品的双向电泳图谱,图谱经ImageMaster2-D Platinu m5.0分析后检测到每张胶平均有365±9个蛋白点,图3是人血清样品与5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物相互作用的双向电泳图谱,每张胶平均有373±13个蛋白点,将对照图谱与样品图谱进行匹配,以P≤0.05作为标准,经ImageMaster2-D Platinum 5.0分析后共确认差异蛋白21个,如图3所示,差异点的编号已在图中显示。
实施例七结果
利用ImageMaster5.0凝胶图像分析软件获得21个差异表达蛋白质点,再通过NCBInr的Blast的检索,确定了16个差异表达蛋白质(7、11、14、19、20号蛋白未鉴定出结果)的名称(见表2),分别为:角蛋白1,血清转铁蛋白异构体1前体,RecName:Full=补体C1r子组件;AltName:Full=补体组件1子组件r;包含:RecName:Full=补体C1r子组件重链;包含:RecName:Full=补体C1r子组件轻链;标志:前体,α-1-抗胰蛋白酶前体,玻连蛋白前体,激肽原-1异构体2前体,触珠蛋白同种型1前原蛋白,血清对氧磷酶/芳基酯酶1前体,角蛋白,I型细胞骨架10,链A,影响淀粉样途径的转甲状腺素蛋白的共价二聚体,角蛋白,II型细胞骨架6B,载脂蛋白C-III前体,链A,脱氧第四纪血红蛋白结构与配体结合在血红素,血清白蛋白前原蛋白,凝溶胶蛋白同种型前蛋白等。
表2 5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑与人血清样品作用后的差异蛋白信息
实施例八5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑的制备
室温下,取叠氮苯(1.0mmol)与对甲氧基苯乙烯(1.1mmol)溶于THF与HMPA的混合溶剂中(10mL,体积比1:1)中,加入SmI2(2.0mmol),搅拌反应2.5小时后(TLC检测原料消失),反应液浓缩后,用乙酸乙酯稀释、依次用水、饱和食盐水洗涤,无水硫酸钠干燥,过滤,浓缩后,经硅胶柱层析得到5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑(淡黄色固体,226mg,收率90.0%,mp100-102℃,1H NMR(CDCl3,400MHz):δ=7.81(s,1H),7.44(d,J=2.8Hz,3H)7.39(s,2H),7.15(d,J=8Hz,2H),6.86(d,J=7.6Hz,2H),3.80(s,3H);13C NMR(CDCl3,100MHz):δ=160.2,137.6,136.7,132.9,129.9,129.3,129.1,125.2,118.9,114.3,55.3)。
室温下,取叠氮苯(1.0mmol)与对甲氧基苯乙烯(1.3mmol)溶于THF与HMPA的混合溶剂中(10mL,体积比1:1)中,加入SmI2(2.0mmol),搅拌反应2.5小时后(TLC检测原料消失),反应液浓缩后,用乙酸乙酯稀释、依次用水、饱和食盐水洗涤,无水硫酸钠干燥,过滤,浓缩后,经硅胶柱层析得到5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑(淡黄色固体,230mg,结构确证数据与报道一致)。
室温下,取叠氮苯(1.0mmol)与对甲氧基苯乙烯(1.3mmol)溶于THF与HMPA的混合溶剂中(10mL,体积比1:1)中,加入SmI2(0.2mmol),搅拌反应2.5小时后,TLC检测显示反应液中反应物仍是主点,隐约可见目标产物点。
Claims (7)
1.一种5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑与血清作用检测差异蛋白的方法,其特征在于包括如下步骤:
(1)将血清蛋白与5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物溶液混合均匀得样品a,恒温培养得样品b;
(2)去除样品b中的高丰度蛋白得样品c;
(3)用Bradford法对样品c的蛋白浓度进行定量;
(4)进行双向电泳实验
①按每胶条蛋白质上样量1.3mg,上样总体积为455uL的标准水化上样,进行第一向等电聚焦;
②第一向等电聚焦完毕后,将胶条平衡后转到聚丙烯酰胺凝胶进行第二垂直电泳实验;
③电泳结束后进行考马斯亮蓝染色,脱色,保存,扫描分析;
(5)胶片图像处理及质谱鉴定
①凝胶图像分析,筛选出表达异常的蛋白点并做上标记;
②差异蛋白点的酶解及质谱鉴定;
(6)结果
利用ImageMaster5.0凝胶图像分析软件获得21个差异表达蛋白质点,再通过NCBInr的Blast的检索,确定了其中1-6、8-10、12-13、15-18、21号差异表达蛋白的信息见下表,
2.权利要求1所述的方法,其特征在于所述步骤(1)中5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物溶液为5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物的甲醇溶液,样品a中5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑化合物的浓度为2.0×10-3mol/L。
3.权利要求1所述的方法,其特征在于所述步骤(1)中恒温培养:温度为37℃,转速为r=280rpm,时间为1h。
4.权利要求1所述的方法,其特征在于所述血清蛋白为人血清蛋白。
5.权利要求1所述的方法,其特征在于所述步骤(2)中去除高丰度蛋白的方法为按照AndyBio试剂盒要求去除高丰度蛋白。
6.权利要求1所述的方法,其特征在于所述步骤(4)中胶条规格为24cm非线性IPG胶条IPG pH=3~10NL。
7.5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑在与血清蛋白作用确定差异蛋白中的应用,其特征在于所述5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑的制备方法包括如下步骤:室温下,将叠氮苯与对甲氧基苯乙烯溶于有机溶剂中,加入SmI2,搅拌反应2.5小时后得到5-(4-甲氧基苯基)-1-苯基-1H-1,2,3-三唑;所述有机溶剂为THF与HMPA体积比为1:1的混合溶液;叠氮苯、对甲氧基苯乙烯、SmI2的摩尔比为1:1.1-1.3:2.0。
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