CN113527459A - 一种萃取剂及其制备方法与应用 - Google Patents

一种萃取剂及其制备方法与应用 Download PDF

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CN113527459A
CN113527459A CN202110716922.2A CN202110716922A CN113527459A CN 113527459 A CN113527459 A CN 113527459A CN 202110716922 A CN202110716922 A CN 202110716922A CN 113527459 A CN113527459 A CN 113527459A
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马菁菁
黄晟鸿
卢传礼
周光雄
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Abstract

本发明公开了一种萃取剂及其制备方法与应用，所述萃取剂包括A液和B液，所述A液为乙腈；所述B液以水为溶剂，包含铵盐。本发明的萃取剂只需通过一步操作即可有效降低阿尔兹海默症检测样本中高丰度蛋白的含量，同时不会造成目标成分损失，高丰度蛋白的去除率至少可以达到98％，样本经过该萃取试剂萃取后，可直接进行LC‑MS/MS定量，不需要其它额外操作步骤。

Description

一种萃取剂及其制备方法与应用
技术领域
本发明属于生物技术领域，具体涉及一种萃取剂及其制备方法与应用。
背景技术
阿尔兹海默症(Alzheimer's disease，AD)是神经系统退行性疾病，伴有特殊病理和生化变化，是一种获得性、持续性及全面性的认知功能障碍的临床综合症。
相关技术表明，β-淀粉样蛋白(Aβ)是淀粉样蛋白前体(APP)经蛋白水解酶作用后的底物，由第21号染色体编码，是阿尔兹海默症的主要病理蛋白之一。APP被至少3种酶加工，其切割途径包括分泌酶途径I和分泌酶途径II。在分泌酶途径中，APP首先被β-分泌酶裂解，随后在γ-分泌酶作用下，被切割形成含有39-43个氨基酸的多肽。其中，Aβ1-42是最常见的亚型之一，由42-43个氨基酸组成的蛋白质片段，主要位于阿尔兹海默症患者的脑内。Aβ1-42多肽与阿尔兹海默症(AD)密切相关，是脑脊液和血浆中的关键生物标志物之一。由于临床样本(脑脊液)中Aβ1-42多肽的浓度极低，约400pg/mL同时容易发生容易聚集，且存在大量其它高丰度蛋白，严重干扰对Aβ1-42多肽的测定，导致对Aβ1-42多肽的检测难度极高，尚不能很好的应用于AD的临床诊断和早期筛查。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出一种萃取剂，能够用于阿尔兹海默症临床样本前处理，有效降低样本中高丰度蛋白含量。
本发明还提出一种具有上述萃取剂的应用。
根据本发明的一个方面，提出了一种萃取剂，包括A液和B液，所述A液为乙腈；所述B液以水为溶剂，包含铵盐。
在本发明的一些实施方式中，所述铵盐为氟化铵、甲酸铵、乙酸铵和碳酸氢铵中的一种。
在本发明的一些实施方式中，所述B液中铵盐的含量为1-50mmol/L。
在本发明的一些实施方式中，所述B液中铵盐的含量为10-30mmol/L。
在本发明的一些实施方式中，所述A液和B液的体积比为40～95：5～60。
本发明的第二方面提出了上述萃取剂的应用，所述萃取剂用于多肽萃取。
在本发明的一些实施方式中，所述萃取剂在制备阿尔兹海默症检测试剂中的应用。
在本发明的一些实施方式中，所述萃取剂用于Aβ1-42淀粉样多肽的萃取。
在本发明的一些实施方式中，所述萃取剂用于阿尔兹海默症样本中高丰度蛋白的去除。
根据本发明的实施方式的一种萃取剂，至少具有以下有益效果：本发明的萃取剂，只需通过一步操作即可有效降低阿尔兹海默症检测样本中高丰度蛋白的含量，同时不会造成目标成分损失，高丰度蛋白的去除率至少可以达到98％，样本经过该萃取试剂萃取后，可直接进行LC-MS/MS定量，不需要其它额外操作步骤。
附图说明
下面结合附图和实施例对本发明做进一步的说明，其中：
图1为本发明测试例中的UPLC-MS/MS对Aβ1-42浓度进行测定的标准曲线图；
图2为本发明测试例中的ACSF蛋白质标准曲线图；
图3为本发明测试例中的采用实施例1-3制备的萃取剂萃取Aβ1-42多肽后样品溶液的UPLC-MS/MS特征图；
图4为本发明测试例中的采用对比例1-2制备的萃取剂萃取Aβ1-42多肽后样品溶液的UPLC-MS/MS特征图。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，均可从商业途径得到的试剂和材料。
用购自南京亿迅生物科技有限公司的人工脑脊液缓冲液配制成含0.15mg/mL白蛋白和0.04mg/mLγ-球蛋白的模拟脑脊液(标记为ACSF)。
实施例1
本实施例制备了一种萃取剂，包括A液和B液，A液为乙腈和B液为水溶液(包含20mmol/L氟化铵)两种试剂按比例混合而成，A液和B液的体积比为90：10。
实施例2
本实施例制备了一种萃取剂，包括A液和B液，A液为乙腈，B液为水溶液(包含20mmol/L甲酸铵)，两种试剂按比例混合而成，A液和B液的体积比为80：20。
实施例3
本实施例制备了一种萃取剂，包括A液和B液，A液为乙腈，B液为水溶液(包含20mmol/L乙酸铵)，两种试剂按比例混合而成，A液和B液的体积比为90：10。
对比例1
本对比例制备了一种萃取剂，包括A液和B液，A液为乙腈，B液为纯水，两种试剂按比例混合而成，A液和B液的体积比为90：10。
对比例2
本对比例制备了一种萃取剂，包括A液和B液，A液为乙腈，B液为水溶液(含0.1％甲酸)，两种试剂按比例混合而成，A液和B液的体积比为90：10。
试验例
1、标准曲线的制备
A、Aβ1-42浓度测定标准曲线的制备
ACSF加入等体积的萃取剂，充分涡旋震荡混匀30s后，冰浴放置30min。12000rpm高速离心5min，取上清液作为溶剂，配制浓度分别为100、200、400、500、800、1000、14000、1600、2000pg/mL的Aβ1-42多肽标准品溶液。
取190μL Aβ1-42多肽标准品溶液，加入10μL 20.0ng/mL[15N53]标记的Aβ1-42多肽溶液，混匀后进行UPLC-MS/MS检测，检测用离子对分别是903.700→886.100和914.500→896.600。记录Aβ1-42和[15N53]标记Aβ1-42的峰面积，计算两个峰面积的比值。
通过最小二乘法建立峰面积比值与Aβ1-42浓度间的线性关系。
结果如图1所示，从图中可以看出，在100-2000pg/mL范围内，Aβ1-42和[15N53]标记Aβ1-42的峰面积比值与Aβ1-42的浓度呈良好的线性关系(R2＝0.9909)。
B、ACSF蛋白质标准曲线的制备
取ACSF(总蛋白含量为0.19mg/mL)，用缓冲液依次稀释成下列浓度38、9.5、2.375、0.594和0.148μg/mL。取0.7mL上述样品溶液于5mL离心管中，加入3.5mL考马斯亮蓝溶液，充分混匀后，放置10min。分光光度法测量反应液在595nm处的吸光度值。1mL水与5mL考马斯亮蓝混匀，放置10min，作为对照组。通过最小二乘法建立反应液吸光度值与总蛋白浓度间的线性关系。
结果如图2所示，从图中可以看出，在0.594-38μg/mL范围内，反应体系在595nm处的吸光度值与总蛋白的浓度呈良好的线性关系(R2＝0.9965)。
2、样品的检测
分别用实施例1-3和对比例1-2制备的多肽萃取剂对样品进行处理，测定实施例1-3和对比例1-2制备的多肽萃取剂对样品中Aβ1-42多肽含量的影响。
向180μL ACSF中加入20μL不同浓度的Aβ1-42多肽标准品溶液，使得样品中Aβ1-42的终浓度依次为0.2、0.4、0.6、0.8、1.0和2.0ng/mL。
上述样品分别加入等体积的Aβ1-42多肽萃取剂(实施例1制备的多肽萃取剂、实施例2制备的多肽萃取剂、实施例3制备的多肽萃取剂、对比例1制备的多肽萃取剂、对比例2制备的多肽萃取剂)，充分涡旋震荡混匀30s后，冰浴放置30min；12000rpm高速离心5min，取190μL上清液，加入10μL 20.0ng/mL[15N53]标记的Aβ1-42多肽溶液，混匀后进行UPLC-MS/MS检测，检测用离子对分别是903.700→886.100和914.500→896.600。记录Aβ1-42和[15N53]标记Aβ1-42的峰面积，计算两个峰面积的比值。并按照Aβ1-42浓度测定标准曲线计算出样品中Aβ1-42的浓度；
取700μL上清液，加入3.5mL考马斯亮蓝工作液，充分混匀后室温下放置10min。利用紫外-可见分光光度计记录反应液在595nm处的吸光度值，并根据ACSF蛋白质标准曲线计算出样品中总蛋白的浓度。
表1实施例1-3测试结果
Figure BDA0003135332260000051
表2对比例1-2测试结果
Figure BDA0003135332260000052
实验结果如表1-2所示，表1为本发明实施例1-3制备的多肽萃取剂对样本中Aβ1-42多肽含量的影响的结果，从表中可以看出，经过萃取剂处理后，样本中总蛋白的含量降低了98％以上，但是Aβ1-42多肽的含量没有明显的降低，Aβ1-42多肽检测的准确度为97.2％～104.2％。
采用实施例1-3制备的萃取剂萃取Aβ1-42多肽后样品溶液的UPLC-MS/MS特征图如图3所示，采用对比例1-2制备的萃取剂萃取Aβ1-42多肽后样品溶液的UPLC-MS/MS特征图如图4所示。从图中可以看出，萃取剂中含有铵盐(氟化铵、甲酸铵、乙酸铵)时，Aβ1-42多肽峰的信号-噪音(S/N)比值较高，能够满足定量要求。而不含有铵盐时，Aβ1-42多肽峰的S/N值明显降低，使得定量准确度降低，尤其在低浓度时，S/N值已经不能够满足定量要求。
表2为对比例1和对比例2制备的萃取试剂对样本中Aβ1-42多肽含量的影响的结果，从表中可以看出，对比例1和对比例2制备的萃取试剂对样本进行处理后，模拟脑脊液中高丰度蛋白的清除率均低于95.5％。对于低浓度Aβ1-42多肽样本(0.2和0.4ng/mL)检测的准确度明显降低，检测的精密度也明显下降。
综上所示，本发明实施例制备的萃取试剂在充分去除样本中高丰度蛋白干扰的前提下，能够充分萃取Aβ1-42多肽，样本前处理仅需要萃取一步骤，可以有效简化操作步骤、减少操作误差。
上面结合附图对本发明实施例作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。

Claims (9)

1.一种萃取剂，其特征在于，包括A液和B液，所述A液为乙腈；所述B液以水为溶剂，包含铵盐。
2.根据权利要求1所述的萃取剂，其特征在于，所述铵盐为氟化铵、甲酸铵、乙酸铵和碳酸氢铵中的一种。
3.根据权利要求1所述的萃取剂，其特征在于，B液中铵盐的含量为1-50mmol/L。
4.根据权利要求3所述的萃取剂，其特征在于，B液中铵盐的含量为10-30mmol/L。
5.根据权利要求1所述的萃取剂，其特征在于，所述A液和所述B液的体积比为40～95：5～60。
6.根据权利要求1-5任一所述的萃取剂在萃取多肽中的应用。
7.根据权利要求1-5任一所述的萃取剂在制备阿尔兹海默症检测试剂中的应用。
8.根据权利要求1-5任一所述的萃取剂在Aβ1-42淀粉样多肽的萃取中的应用。
9.根据权利要求1-5任一所述的萃取剂在去除阿尔兹海默症样本中高丰度蛋白中的应用。
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