MSACL 2018 US Abstract


Notice: Undefined variable: keynote in /home/msacl0017/public_html/view_abstract/MSACL_2018_US.php on line 84

Topic: Metabolomics

High-Throughput Screening of Enzymatic Reactions Using IR-MALDESI

Mans Ekelof (Presenter)
North Carolina State University

Bio: Måns Ekelöf received his master’s degree in chemistry and chemical engineering from KTH, Stockholm, Sweden in 2013. He is currently a third-year graduate student in the Muddiman group at NC State University, working on the development of new methods to generate ions for direct mass spectrometric analysis of complex samples.

Authorship: Mans Ekelof (1), Milad Nazari (1), Sitora Khodjaniyazova (1), Nathaniel L. Elsen (2), Jon D. Williams (2), David C. Muddiman (1)
(1) North Carolina State University, Raleigh, NC 27695, USA, (2) AbbVie Inc., North Chicago, IL 60064, USA

Short Abstract

The ability to rapidly and accurately characterize samples is of critical importance in the drug discovery process as well as in the clinical testing laboratory. For many screens and assays, the benefits of mass spectrometric analysis are outweighed by sample compatibility concerns and low throughput. We present a method for high-throughput measurement of analytes in the IDH1-catalyzed conversion of isocitric acid to 2-oxoglutaric acid, sampled directly from the reaction vessel with no sample treatment, using infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) coupled to a Q Exactive Plus mass spectrometer.

Long Abstract

Introduction

Despite the rapid spread of mass spectrometry in virtually all fields of analytical chemistry, optical spectroscopy remains the method of choice for many high-throughput screening applications. Using automated absorbance or fluorescence spectroscopy instruments, it is possible to analyze hundreds of samples per second with acceptable hit rates. The universality and label-free specificity of mass spectrometry would be ideal in a high-throughput instrument, but the need for sample preparation and/or chromatographic separation adds complexity and limits the speed of the analysis.

In the presented IR-MALDESI approach [1] to high-throughput sampling and ionization, a mid-IR laser is used to aerosolize reaction solution directly from the wells of a commercial microtiter plate. Analytes are desorbed and ionized from the ejected particles through interactions with an intersecting electrospray plume. This electrospray post-ionization approach has been proven to have significant tolerance for contaminants such as buffer salts and surfactants, requiring no cleanup before sampling and ionization. Since the laser sampling procedure requires only a few milliseconds, this strategy is currently rate-limited only by the duty cycle of the mass spectrometer.

Methods

The IDH1-catalyzed conversion of isocitric acid to 2-oxoglutaric acid was chosen as a model system for high-throughput screening. Reactions were started by addition of 1 nM IDH1 to a mixture of 200 µM of isocitric acid and 80 µM of the cofactor NADP+. All reactions were carried out at room temperature in a buffer system composed of 20 mM tris HCl, 1 mM MgCl2, 1 mM DTT and 0.01% BSA (w/v). The reaction solution was directly analyzed with ten scans at several time points over the course of two hours using IR-MALDESI, and tracked based on the observed conversions of isocitric acid to 2-oxoglutaric acid and NADP+ to NADPH by full MS scans, in the mass ranges of 100-400 and 250-1000 Th, respectively.

Results

The reactants, products and cofactors of the model reaction were found to be readily detectable in the concentration regime and solvent conditions of the experiment. All measurements were taken in wide mass range full MS scans, and reaction completion was tracked as percent conversion. Using measurements at zero and 90 minutes as negative and positive controls, the Z-score [2] of the assay was calculated to 0.65. Eventually, in a simulated single-blind screen, the developed method provided correct identification of all samples.

Conclusions & Discussion

In this pilot study, the method was found to work remarkably well, providing robust hit/miss style semi-quantitative performance at a rate of one sample well per four seconds. The combination of direct laser sampling with electrospray post-ionization makes IR-MALDESI highly sample agnostic, requiring no particular changes to the sample preparation workflows in the lab. Current efforts are focused on developing and evaluating a truly high throughput (>1 Hz) quantitative assay for enzymatic reactions, using internal standard addition to maximize assay quality.


References & Acknowledgements:

[1] Nazari M, Ekelöf M et al., Rapid Commun. Mass Spectrom., In Press

[2] J. Zhang, T. D. Y. Chung, K. R. Oldenburg., J. Biomol. Screen. 1999


Financial Disclosure

DescriptionY/NSource
Grantsno
Salaryno
Board Memberno
Stockno
Expensesno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

no