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A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1.
- Source :
-
Analyst . 6/7/2020, Vol. 145 Issue 11, p3899-3908. 10p. - Publication Year :
- 2020
-
Abstract
- The enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the conversion of isocitrate to alpha-ketoglutarate (αKG) and has emerged as an important therapeutic target for glioblastoma multiforme (GBM). Current methods for assaying IDH1 remain poorly suited for high-throughput screening of IDH1 antagonists. This paper describes a high-throughput and quantitative assay for IDH1 that is based on the self-assembled monolayers for matrix-assisted laser desorption/ionization-mass spectrometry (SAMDI-MS) method. The assay uses a self-assembled monolayer presenting a hydrazide group that covalently captures the αKG product of IDH1, where it can then be detected by MALDI-TOF mass spectrometry. Co-capture of an isotopically-labeled αKG internal standard allows the αKG concentration to be quantitated. The assay was used to analyze a series of standard αKG solutions and produced minimal error in measured αKG concentration values. The suitability of the assay for high-throughput analysis was evaluated in a 384-sample biochemical IDH1 screen. Cells expressing IDH1 were lysed and the lysate was applied to the monolayer to capture αKG, which was then quantitated using the SAMDI-MS assay. Cells in which IDH1 expression was reduced by small-interfering RNA exhibited a corresponding decrease in αKG concentration as measured by the assay. Application of the assay toward the high-throughput screening of IDH1 inhibitors or knockdown agents may facilitate the discovery of treatments for GBM. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00032654
- Volume :
- 145
- Issue :
- 11
- Database :
- Academic Search Index
- Journal :
- Analyst
- Publication Type :
- Academic Journal
- Accession number :
- 143550808
- Full Text :
- https://doi.org/10.1039/d0an00174k