Advertisement

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

  • Markéta Machálková
  • Jan Schejbal
  • Zdeněk Glatz
  • Jan Preisler
Research Paper

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.

Graphical abstract

Keywords

β-Secretase Enzyme assay High throughput Inhibition MALDI TOF MS 

Abbreviations

ACN

Acetonitrile

AcOH

Acetic acid

AcONa

Sodium acetate

AcONH4

Ammonium acetate

AD

Alzheimer’s disease

APP

Amyloid precursor protein

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

IC50

Half-maximal inhibitory concentration

KM

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

Notes

Funding information

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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

216_2018_1354_MOESM1_ESM.pdf (449 kb)
ESM 1 (PDF 448 kb)

References

  1. 1.
    Dementia. World Health Organization. http://www.who.int/mediacentre/factsheets/fs362/en/. 2017. Accessed 2 March 2018.
  2. 2.
    Glenner G, Wong C. Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun. 1984;120(3):885–90.  https://doi.org/10.1016/S0006-291X(84)80190-4.CrossRefPubMedGoogle Scholar
  3. 3.
    Lacor PN, Buniel MC, Furlow PW, Sanz Clemente A, Velasco PT, Wood M, et al. Aβ oligomer-induced aberrations in synapse composition, shape, and density provide a molecular basis for loss of connectivity in Alzheimer’s disease. J Neurosci. 2007;27(4):796–807.  https://doi.org/10.1523/jneurosci.3501-06.2007. CrossRefPubMedGoogle Scholar
  4. 4.
    Luo Y, Bolon B, Kahn S, Bennett BD, Babu-Khan S, Denis P, et al. Mice deficient in BACE1, the Alzheimer’s β-secretase, have normal phenotype and abolished β-amyloid generation. Nat Neurosci. 2001;4:231.CrossRefGoogle Scholar
  5. 5.
    Cole SL, Vassar R. The Alzheimer’s disease β-secretase enzyme, BACE1. Mol Neurodegener. 2007;2(1):22.CrossRefGoogle Scholar
  6. 6.
    Mancini F, De Simone A, Andrisano V. Beta-secretase as a target for Alzheimer’s disease drug discovery: an overview of in vitro methods for characterization of inhibitors. Anal Bioanal Chem. 2011;400(7):1979–96.CrossRefGoogle Scholar
  7. 7.
    Grüninger-Leitch F, Schlatter D, Küng E, Nelböck P, Döbeli H. Substrate and inhibitor profile of BACE (β-secretase) and comparison with other mammalian aspartic proteases. J Biol Chem. 2002;277(7):4687–93.CrossRefGoogle Scholar
  8. 8.
    Mancini F, Naldi M, Cavrini V, Andrisano V. Multiwell fluorometric and colorimetric microassays for the evaluation of beta-secretase (BACE-1) inhibitors. Anal Bioanal Chem. 2007;388(5–6):1175–83.CrossRefGoogle Scholar
  9. 9.
    Mancini F, Andrisano V. Development of a liquid chromatographic system with fluorescent detection for β-secretase immobilized enzyme reactor on-line enzymatic studies. J Pharm Biomed Anal. 2010;52(3):355–61.CrossRefGoogle Scholar
  10. 10.
    Yi X, Hao Y, Xia N, Wang J, Quintero M, Li D, et al. Sensitive and continuous screening of inhibitors of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) at single SPR chips. Anal Chem. 2013;85(7):3660–6.CrossRefGoogle Scholar
  11. 11.
    Liu R, Liu Y-C, Meng J, Zhu H. Zhang X. A microfluidics-based mobility shift assay to identify new inhibitors of β-secretase for Alzheimer’s disease. Anal Bioanal Chem. 2017;409(28):6635–42.CrossRefGoogle Scholar
  12. 12.
    Schejbal J, Slezáčková L, Řemínek R, Glatz Z. A capillary electrophoresis-mass spectrometry based method for the screening of β-secretase inhibitors as potential Alzheimer’s disease therapeutics. J Chromatogr A. 2017;1487:235–41.CrossRefGoogle Scholar
  13. 13.
    Greis K. Mass spectrometry for enzyme assays and inhibitor screening: an emerging application in pharmaceutical research. Mass Spectrom Rev. 2007;26(3):324–39.  https://doi.org/10.1002/mas.20127.CrossRefPubMedGoogle Scholar
  14. 14.
    Duncan MW, Roder H, Hunsucker SW. Quantitative matrix-assisted laser desorption/ionization mass spectrometry. Brief Funct Genomic Proteomic. 2008;7(5):355–70.CrossRefGoogle Scholar
  15. 15.
    Kang M, Tholey A, Heinzle E. Application of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the measurement of enzyme activities. Rapid Commun Mass Spectrom. 2001;15(15):1327–33.  https://doi.org/10.1002/rcm.376.CrossRefPubMedGoogle Scholar
  16. 16.
    Bungert D, Heinzle E, Tholey A. Quantitative matrix-assisted laser desorption/ionization mass spectrometry for the determination of enzyme activities. Anal Biochem. 2004;326(2):167–75.  https://doi.org/10.1016/j.ab.2003.11.013.CrossRefPubMedGoogle Scholar
  17. 17.
    Ritorto M, Ewan R, Perez-Oliva A, Knebel A, Buhrlage S, Wightman M, et al. Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. Nat Commun. 2014;5  https://doi.org/10.1038/ncomms5763.
  18. 18.
    Greis K, Zhou S, Burt T, Carr A, Dolan E, Easwaran V, et al. MALDI-TOF MS as a label-free approach to rapid inhibitor screening. J Am Soc Mass Spectrom. 2006;17(6):815–22.  https://doi.org/10.1016/j.jasms.2006.02.019.CrossRefPubMedGoogle Scholar
  19. 19.
    Guitot K, Scarabelli S, Drujon T, Bolbach G, Amoura M, Burlina F, et al. Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Biochem. 2014;456:25–31.  https://doi.org/10.1016/j.ab.2014.04.006.CrossRefPubMedGoogle Scholar
  20. 20.
    Patrie S, Roth M, Plymire D, Maresh E, Zhang J. Measurement of blood protease kinetic parameters with self-assembled mono layer ligand binding assays and label-free MALDI-TOF MS. Anal Chem. 2013;85(21):10597–604.  https://doi.org/10.1021/ac402739z.CrossRefPubMedGoogle Scholar
  21. 21.
    Beeman K, Baumgartner J, Laubenheimer M, Hergesell K, Hoffmann M, Pehl U, et al. Integration of an in situ MALDI-based high-throughput screening process: a case study with receptor tyrosine kinase c-MET. SLAS Discov. 2017;22(10):1203–10.  https://doi.org/10.1177/2472555217727701.CrossRefPubMedGoogle Scholar
  22. 22.
    Guitot K, Drujon T, Burlina F, Sagan S, Beaupierre S, Pamlard O, et al. A direct label-free MALDI-TOF mass spectrometry based assay for the characterization of inhibitors of protein lysine methyltransferases. Anal Bioanal Chem. 2017;409(15):3767–77.CrossRefGoogle Scholar
  23. 23.
    Zovo K, Helk E, Karafin A, Tõugu V, Palumaa P. Label-free high-throughput screening assay for inhibitors of Alzheimer’s amyloid-β peptide aggregation based on MALDI MS. Anal Chem. 2010;82(20):8558–65.CrossRefGoogle Scholar
  24. 24.
    Van Loco J, Elskens M, Croux C, Beernaert H. Linearity of calibration curves: use and misuse of the correlation coefficient. Accred Qual Assur. 2002;7(7):281–5.CrossRefGoogle Scholar
  25. 25.
    Leite J, Hajivandi M, Diller T, Pope R. Removal of sodium and potassium adducts using a matrix additive during matrix-associated laser desorption/ionization time-of-flight mass spectrometric analysis of peptides. Rapid Commun Mass Spectrom. 2004;18(23):2953–9.  https://doi.org/10.1002/rcm.1711.CrossRefPubMedGoogle Scholar
  26. 26.
    Beavis R, Chait B. Matrix-assisted laser desorption ionization mass-spectrometry of proteins. High Resolution Separation and Analysis of Biological Macromolecules, Pt A. 1996;270:519–51.Google Scholar
  27. 27.
    Řemínek R, Slezáčková L, Schejbal J, Glatz Z. Development and comprehensive comparison of two on-line capillary electrophoretic methods for β-secretase inhibitor screening. J Chromatogr A. 2017;1518:89–96.CrossRefGoogle Scholar
  28. 28.
    May PC, Willis BA, Lowe SL, Dean RA, Monk SA, Cocke PJ, et al. The potent BACE1 inhibitor LY2886721 elicits robust central Aβ pharmacodynamic responses in mice, dogs, and humans. J Neurosci. 2015;35(3):1199–210.CrossRefGoogle Scholar
  29. 29.
    May P, Boggs L, Brier R, Calligaro D, Citron M, Day T, et al. Preclinical characterization of LY2886721: a BACE1 inhibitor in clinical development for early Alzheimer’s disease. Alzheimers Dement. 2012;8(4):704–5.  https://doi.org/10.1016/j.jalz.2012.05.235. CrossRefGoogle Scholar
  30. 30.
    Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, et al. Purification and cloning of amyloid precursor protein β-secretase from human brain. Nature. 1999;402(6761):537.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Markéta Machálková
    • 1
    • 2
  • Jan Schejbal
    • 3
  • Zdeněk Glatz
    • 3
  • Jan Preisler
    • 1
    • 2
  1. 1.Department of Analytical Chemistry, Faculty of ScienceMasaryk UniversityBrnoCzech Republic
  2. 2.Central European Institute of Technology (CEITEC)Masaryk UniversityBrnoCzech Republic
  3. 3.Department of Biochemistry, Faculty of ScienceMasaryk UniversityBrnoCzech Republic

Personalised recommendations