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A label-free MALDI TOF MS-based method for studying the kinetics and inhibitor screening of the Alzheimer’s disease drug target β-secretase

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Abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities including sensitivity, minute sample consumption, and label-free detection, all of which are highly desired in enzyme assays. On the other hand, the application of MALDI TOF MS is usually limited by high concentrations of MS-incompatible compounds in the reaction mixture such as salts or organic solvents. Here, we introduce kinetic and inhibition studies of β-secretase (BACE1), a key enzyme of the progression of Alzheimer’s disease. Compatibility of the enzyme assay with MALDI TOF MS was achieved, providing both a complex protocol including a desalting step designed for rigorous kinetic studies and a simple mix-and-measure protocol designed for high-throughput inhibitor screening. In comparison with fluorescent or colorimetric assays, MALDI TOF MS represents a sensitive, fast, and label-free technique with minimal sample preparation. In contrast to other MS-based methodological approaches typically used in drug discovery processes, such as a direct injection MS or MS-coupled liquid chromatography or capillary electrophoresis, MALDI TOF MS enables direct analysis and is a highly suitable approach for high-throughput screening. The method’s applicability is strongly supported by the high correlation of the acquired kinetic and inhibition parameters with data from the literature as well as from our previous research.

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Abbreviations

ACN:

Acetonitrile

AcOH:

Acetic acid

AcONa:

Sodium acetate

AcONH4 :

Ammonium acetate

AD:

Alzheimer’s disease

APP:

Amyloid precursor protein

Aβ:

Amyloid β peptide

BACE1:

β-secretase

CE-ESI MS:

Capillary electrophoresis coupled to mass spectrometry with electrospray ionization

CHCA:

α-cyano-4-hydroxycinnamic acid

DMSO:

Dimethyl sulfoxide

ESI:

Electrospray ionization

FRET:

Fluorescence resonance energy transfer

IB:

Incubation buffer

IC 50 :

Half-maximal inhibitory concentration

K M :

Michaelis constant

MALDI MS:

Matrix-assisted laser desorption/ionization mass spectrometry

MALDI TOF MS:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

MS:

Mass spectrometry

QTOF:

Quadrupole time-of-flight

S/N :

Signal-to-noise ratio

TFA:

Trifluoracetic acid

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Funding

This work was supported by the Czech Science Foundation (GA16-06106S), the Grant Agency of Masaryk University (MUNI/G/0974/2016), and the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601).

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Authors and Affiliations

  1. Department of Analytical Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic

    Markéta Machálková & Jan Preisler

  2. Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic

    Markéta Machálková & Jan Preisler

  3. Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic

    Jan Schejbal & Zdeněk Glatz

Authors
  1. Markéta Machálková
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  2. Jan Schejbal
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  3. Zdeněk Glatz
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  4. Jan Preisler
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Corresponding author

Correspondence to Jan Preisler.

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The authors declare that they have no conflicts of interest.

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Machálková, M., Schejbal, J., Glatz, Z. et al. A label-free MALDI TOF MS-based method for studying the kinetics and inhibitor screening of the Alzheimer’s disease drug target β-secretase. Anal Bioanal Chem 410, 7441–7448 (2018). https://doi.org/10.1007/s00216-018-1354-6

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  • DOI: https://doi.org/10.1007/s00216-018-1354-6

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