Issue 11, 2020

A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1

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.

Graphical abstract: A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1

Supplementary files

Article information

Article type
Paper
Submitted
22 1 2020
Accepted
08 4 2020
First published
09 4 2020

Analyst, 2020,145, 3899-3908

A high-throughput SAMDI-mass spectrometry assay for isocitrate dehydrogenase 1

S. E. Anderson, N. S. Fahey, J. Park, P. T. O'Kane, C. A. Mirkin and M. Mrksich, Analyst, 2020, 145, 3899 DOI: 10.1039/D0AN00174K

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements