Daniel Carvalho, Bruna Barbosa Laurentino, Camila Loreta Rocha, J. Kool, G. Somsen, Erika R Amstalden van Hove, C. Cardoso
{"title":"基于固定化酶钾激肽和质谱法的活性测定","authors":"Daniel Carvalho, Bruna Barbosa Laurentino, Camila Loreta Rocha, J. Kool, G. Somsen, Erika R Amstalden van Hove, C. Cardoso","doi":"10.3389/frans.2022.1018115","DOIUrl":null,"url":null,"abstract":"Deregulated activity and expression of human kallikreins (KLKs) may be involved in various pathologies, so these enzymes are an attractive biological target for identifying molecules that can modulate KLK activity. This identification involves applying fast and efficient screening methods. This work describes an off-line assay with mass spectrometry (MS) detection that uses KLK immobilized on Sepharose-NHS as a micro-column configuration (IMER-KLK-Sepharose-NHS). The mass spectrometry used has an ion trap analyzer and electrospray ionization (EIS). The HPLC-MS method for quantifying KLK activity was developed. The enzymatic assay conditions were optimized, and the IMER-KLK-Sepharose-NHS kinetic parameter (KMapp = 15.48 ± 3 μmol L−1) was evaluated. Finally, the method was validated by using leupeptin as a reference inhibitor (IC50 = 0.85 ± 0.10 μmol L−1). The developed method was able to identify the reference inhibitor and can be an alternative for screening KLK inhibitors.","PeriodicalId":73063,"journal":{"name":"Frontiers in analytical science","volume":" ","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2022-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Activity assay based on the immobilized enzyme kallikrein and mass spectrometry\",\"authors\":\"Daniel Carvalho, Bruna Barbosa Laurentino, Camila Loreta Rocha, J. Kool, G. Somsen, Erika R Amstalden van Hove, C. Cardoso\",\"doi\":\"10.3389/frans.2022.1018115\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Deregulated activity and expression of human kallikreins (KLKs) may be involved in various pathologies, so these enzymes are an attractive biological target for identifying molecules that can modulate KLK activity. This identification involves applying fast and efficient screening methods. This work describes an off-line assay with mass spectrometry (MS) detection that uses KLK immobilized on Sepharose-NHS as a micro-column configuration (IMER-KLK-Sepharose-NHS). The mass spectrometry used has an ion trap analyzer and electrospray ionization (EIS). The HPLC-MS method for quantifying KLK activity was developed. The enzymatic assay conditions were optimized, and the IMER-KLK-Sepharose-NHS kinetic parameter (KMapp = 15.48 ± 3 μmol L−1) was evaluated. Finally, the method was validated by using leupeptin as a reference inhibitor (IC50 = 0.85 ± 0.10 μmol L−1). The developed method was able to identify the reference inhibitor and can be an alternative for screening KLK inhibitors.\",\"PeriodicalId\":73063,\"journal\":{\"name\":\"Frontiers in analytical science\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2022-10-19\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Frontiers in analytical science\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.3389/frans.2022.1018115\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in analytical science","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3389/frans.2022.1018115","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Activity assay based on the immobilized enzyme kallikrein and mass spectrometry
Deregulated activity and expression of human kallikreins (KLKs) may be involved in various pathologies, so these enzymes are an attractive biological target for identifying molecules that can modulate KLK activity. This identification involves applying fast and efficient screening methods. This work describes an off-line assay with mass spectrometry (MS) detection that uses KLK immobilized on Sepharose-NHS as a micro-column configuration (IMER-KLK-Sepharose-NHS). The mass spectrometry used has an ion trap analyzer and electrospray ionization (EIS). The HPLC-MS method for quantifying KLK activity was developed. The enzymatic assay conditions were optimized, and the IMER-KLK-Sepharose-NHS kinetic parameter (KMapp = 15.48 ± 3 μmol L−1) was evaluated. Finally, the method was validated by using leupeptin as a reference inhibitor (IC50 = 0.85 ± 0.10 μmol L−1). The developed method was able to identify the reference inhibitor and can be an alternative for screening KLK inhibitors.