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High-throughput Screening of Ligands towards Targeted Proteins using Native Mass Spectrometry   

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摘要:原位质谱(native mass spectrometry)技术是于近十年发展起来的生物质谱新方法,如今已逐渐成为研究生物大分子蛋白质复合物、化学合成的金属纳米团簇、化学反应的自组装体系、以及蛋白质和其它分子相互作用的重要工具。原位质谱法通常使用接近生理pH的挥发性缓冲盐水溶液以及较温和的温度和电压,在从溶液转移到气相进行质谱分析时保留非共价相互作用和四维蛋白质结构,这使得研究蛋白质-蛋白质相互作用、蛋白质-配体相互作用、亚基结构和化学计量学成为可能。原位质谱技术用于高通量药物筛选的工作原理是将已知起始浓度的蛋白与药物分子孵育后在其非变性条件下直接喷射到质谱仪中,通过游离蛋白、蛋白和药物分子复合物的质谱峰响应强度来计算出蛋白和配体分子的结合常数。

关键词: 原位质谱, 响应强度, 结合常数

材料与试剂

  1. 2 mL进样瓶(Waters,catalog number: 186000307C)
  2. 直喷进样针(Waters,catalog number: M956231AD1-S)
  3. 微量上样针(Eppendorf,catalog number: 5242956003)
  4. 缓冲液交换小柱(BIO-RAD,catalog number: 732-6221)
  5. BCA蛋白浓度测定试剂盒(Beyotime,catalog number: P0012S)
  6. 50 mM 乙酸铵溶液 (见溶液配方)

仪器设备

  1. 离心机(Eppendorf, centrifuge 5810 R)
  2. 多功能酶标仪(BioTek, CytationTM 5型)
  3. 质谱仪(Waters SYNAPT G2-Si)

实验步骤

  1. 样品准备
    1.1
    将靶标蛋白样品溶解在50 mM乙酸铵溶液中;
    1.2
    BCA法测定上述溶液中蛋白浓度,多功能酶标仪测定562 nm的吸光度(He, 2011);
    1.3
    用50 mM 乙酸铵溶液配制一系列不同浓度的药物分子溶液(5 µM、10 µM、15 µM、20 µM、25 µM、30 µM、35 µM、40 µM) (Gavriilidou et al., 2015; Keener et al., 2021);
    1.4
    将靶标蛋白分别与不同浓度的药物分子孵育,轻轻拍打试剂管混匀,室温孵育5 min,10,000 x g离心2 min;

  2. 质谱检测
    2.1
    吸取10 µL样品到直喷进样针中;
    2.2
    将喷针放入到离子源中,如图1所示;
    2.3
    调整喷针位置;
    2.4
    调整仪器参数,使质谱信号达到最强。喷雾电压一般在1.2 kV,锥孔电压40 V,离子源温度30 °C,resolution模式,调整质量范围500-10,000 m/z, 一般采集时间3-5 min;


    图1. 质谱喷针安装图

  3. 数据分析
    3.1
    谱图分析采用Waters公司的MassLynx 4.1软件;
    3.2
    通过如下的公式计算药物分子的结合常数Ka (Liu et al., 2021; Tao et al., 2021)。 R值是通过全部电荷的蛋白和药物分子复合物的质谱峰响应值除以所有电荷的游离蛋白的质谱峰响应值获得;[L]0代表药物分子的起始浓度值,[P]0代表靶蛋白样品的起始浓度值。

可能遇到的问题及调整建议
  1. 蛋白浓度以不超过20 µM为宜,不宜过高,否则容易堵针。
  2. 若蛋白样品初始缓冲液是其他缓冲盐溶液,可以先用3 kD的超滤管截留,用50 mM乙酸铵溶液多次清洗回收。回收的样品进一步使用缓冲液交换柱进行缓冲液交换,尽可能去除与蛋白结合的其他盐离子;
  3. 若质谱采集数据时无信号,可适当通过适当调整电压和氮气辅助加压(视频1)。

    视频1

溶液配方

  1. 50 mM 乙酸铵溶液
    称取192.7 mg 乙酸铵固体于烧杯中,加入ddH2O至50 mL。待完全溶解后,过0.22 µm滤器至已灭菌的玻璃瓶中。

致谢

感谢清华大学蛋白质研究技术中心蛋白质化学与组学平台各位老师对本实验方法的帮助和改进。

参考文献

  1. He, F. L. (2011). BCA (Bicinchoninic Acid) Protein Assay. Bio-101: e44. DOI: 10.21769/BioProtoc.44.
  2. Gavriilidou, A. F. M., Gulbakan, B. and Zenobi, R. (2015). Influence of ammonium acetate concentration on receptor ligand binding affinities measured by native nano ESI-MS: a systematic study.Anal Chem 87 (20): 10378-10384.
  3. Keener, J. E., Zhang, G. Z. and Marty, M. T. (2021). Native mass spectrometry of membrane proteins.Anal Chem 93 (1): 583-597.
  4. Liu, L., Kitova, E. N. and Klassen, J. S. (2011). Quantifying protein-fatty acid interactions using electrospray ionization mass spectrometry. J Am Soc Mass Spectrom 22 (2): 310-318
  5. Tao, Y., Yan, J. Z. and Cai, B. C. (2021). Label-free bio-affinity mass spectrometry for screening and locating bioactive molecules. Mass spectrom Rev 40 (1): 53-71.
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Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:孟献斌, 晏顶霏, 邓海腾. (2022). 利用原位质谱法进行药物靶标蛋白配体的高通量筛选. // 高通量筛选实验手册. Bio-101: e1010886. DOI: 10.21769/BioProtoc.1010886.
How to cite: Meng, X. B., Yan, D. F. and Deng, H. T. (2022). High-throughput Screening of Ligands towards Targeted Proteins using Native Mass Spectrometry. // High-throughput Screening Protocol eBook. Bio-101: e1010886. DOI: 10.21769/BioProtoc.1010886.
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