Obi Naoko, Fukuda Tetsuya, Nakayama Noboru, Ervin John, Bando Yasuhiko, Nishimura Toshihide, Nagatoishi Satoru, Tsumoto Kouhei, Kawamura Takeshi
Nissha Co. Ltd, Kyoto, Japan.
Biosys Technologies Inc., Tokyo, Japan.
Rapid Commun Mass Spectrom. 2018 Apr 30;32(8):665-671. doi: 10.1002/rcm.8083.
Drug discovery studies invariably require qualitative and quantitative analyses of target compounds at every stage of drug discovery. We have developed a system combining molecular interaction analysis and mass spectrometry (LC-MS) using the principle of nanopore optical interferometry (nPOI) called molecular interaction kinetics-mass spectrometry (MIK-MS). Since nPOI has high binding capacity, the bond-dissociated compound can be directly detected using LC-MS. In this study, we use carbonic anhydrase II (CAII) as a ligand and apply six small compounds as analytes and report the affinity analysis using MIK-MS.
CAII was immobilized onto a COOH sensor chip using standard amine coupling. A reference surface was prepared by activating and subsequently blocking the surface under identical conditions. An amount of 50 μL of mix solution was injected over the reference channel and sample channel for CAII immobilization. The solutions eluting from the sensor chip were collected from the waste-line of the SKi Pro system every 30 s. Reconstructed elution samples were then injected into the LC-MS/MS system.
A mixture containing furosemide, acetazolamide, 4-sulfamoylbenzoic acid, 5-(dimethylamino)-1-naphthalene sulfonamide (DNSA), sulfanilamide and sulpiride (15 μM each) was injected into the CAII-immobilized sensor chip, and the fractions eluted from the SKi Pro system were collected and subjected to selected reaction monitoring LC-MS characterization. Specific results were obtained for acetazolamide, DNSA, furosemide and sulpiride. The results suggest that the association-dissociation curve of a mixed sample can be obtained by one-time MIK-MS analysis.
Six small-molecule binders of CAII were analyzed quantitatively using nPOI and MIK-MS, and the results were compared to published surface plasmon resonance (SPR) results. The nPOI and SPR results show good agreement, confirming the reliability of the analysis. Time-dependent binding results may be obtained by our MS sensorgram approach. Drugs that meet medical needs in a short period are required; this nPOI-LC-MS system is considered an important tool for rapid drug discovery.
药物发现研究在药物发现的每个阶段都始终需要对目标化合物进行定性和定量分析。我们利用纳米孔光学干涉测量法（nPOI）原理开发了一种结合分子相互作用分析和质谱（LC-MS）的系统，称为分子相互作用动力学-质谱（MIK-MS）。由于nPOI具有高结合能力，键解离的化合物可使用LC-MS直接检测。在本研究中，我们使用碳酸酐酶II（CAII）作为配体，并应用六种小分子化合物作为分析物，报告使用MIK-MS进行的亲和力分析。
使用标准胺偶联法将CAII固定在COOH传感器芯片上。通过在相同条件下活化并随后封闭表面来制备参考表面。将50μL混合溶液注入参考通道和样品通道以固定CAII。每30秒从SKi Pro系统的废液管线收集从传感器芯片洗脱的溶液。然后将重构的洗脱样品注入LC-MS/MS系统。
将含有速尿、乙酰唑胺、4-氨磺酰苯甲酸、5-（二甲基氨基）-1-萘磺酰胺（DNSA）、磺胺和舒必利（各15μM）的混合物注入固定有CAII的传感器芯片中，收集从SKi Pro系统洗脱的馏分，并进行选择反应监测LC-MS表征。获得了乙酰唑胺、DNSA、速尿和舒必利的具体结果。结果表明，通过一次MIK-MS分析可获得混合样品的缔合-解离曲线。
使用nPOI和MIK-MS对六种CAII小分子结合剂进行了定量分析，并将结果与已发表的表面等离子体共振（SPR）结果进行了比较。nPOI和SPR结果显示出良好的一致性，证实了分析的可靠性。通过我们的质谱传感图方法可以获得时间依赖性结合结果。需要在短时间内满足医疗需求的药物；这种nPOI-LC-MS系统被认为是快速药物发现的重要工具。
