Mass spectrometry and immobilized enzymes for the screening of inhibitor libraries
Mass spectrometry and immobilized enzymes for the screening of inhibitor libraries
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DOI：
10.1073/pnas.220403997
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发表时间：
2000-10-24
影响因子：
11.1
通讯作者：
Leary, JA
中科院分区：
文献类型：
--
作者：
Cancilla, MT;Leavell, MD;Leary, JA
利用固定化酶技术和电喷雾离子回旋共振质谱技术，从复杂的混合物中快速筛选酶抑制剂候选物。首先将库混合物喷入质谱仪，并鉴定化合物。随后通过使用过量的酶在正确的条件（缓冲液、pH、温度）下将文库与固定化的感兴趣的酶一起孵育，以确保配体结合的位点过剩。将固定化酶/抑制剂混合物离心，并再次通过电喷雾电离质谱法分析上清液的等分试样。通过比较与固定化酶孵育前后的光谱，快速鉴定潜在的抑制剂。非抑制剂在孵育后显示离子强度无变化，而弱抑制剂显示离子丰度明显降低。一旦识别出抑制剂候选物，将文库重新注入质谱仪中，并根据需要使用串联质谱法来确定抑制剂候选物的结构。这种方法已被成功地证明，通过确定抑制剂的酶胃蛋白酶和谷胱甘肽S-转移酶从19-和17-组分库，分别。进一步表明，固定化酶可以再循环并重新用于连续筛选额外的新文库而无需添加额外的酶。
A technique has been developed to rapidly screen enzyme inhibitor candidates from complex mixtures, such as those created by combinatorial synthesis, Inhibitor libraries are screened by using immobilized enzyme technologies and electrospray ionization ion cyclotron resonance mass spectrometry. The library mixture is first sprayed into the mass spectrometer, and compounds are identified. The library is subsequently incubated with the immobilized enzyme of interest under the correct conditions (buffer, pH, temperature) by using an excess of enzyme to ensure a surplus of sites for ligand binding. The immobilized enzyme/inhibitor mixture is centrifuged, and an aliquot of supernatant is again analyzed by electrospray ionization mass spectrometry. Potential inhibitors are quickly identified by comparison of the spectra before and after incubation with the immobilized enzyme. Non-inhibitors show no change in ion intensity after incubation, whereas weak inhibitors exhibit a visible decrease in ion abundance. Once inhibitor candidates have been identified, the library is reinjected into the mass spectrometer, and tandem mass spectrometry is used to determine the structure of the inhibitor candidates as needed. This method has been successfully demonstrated by identifying inhibitors of the enzymes pepsin and glutathione S-transferase from a 19- and 17-component library, respectively. It is further shown that the immobilized enzyme can be recycled and reused for continuous screening,of additional new libraries without adding additional enzyme.