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Comparative Activity-Based Flavin-Dependent Oxidase Profiling

  • Joanna Krysiak
  • Rolf BreinbauerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1491)

Abstract

Activity-based protein profiling (ABPP) has become a powerful chemoproteomic technology allowing for the dissection of complex ligand–protein interactions in their native cellular environment. One of the biggest challenges for ABPP is the extension of the proteome coverage. In this chapter a new ABPP strategy dedicated to monoamine oxidases (MAO) is presented. These enzymes are representative examples of flavin-dependent oxidases, playing a crucial role in the regulation of nervous system signaling.

Key words

Activity-based protein profiling Click chemistry Flavoproteins Mitsunobu reaction Monoamine oxidase Oxidoreductase Pargyline Target identification 

Abbreviations

ABPP

Activity-based protein profiling

CC

Click chemistry

DMSO

Dimethyl sulfoxide

FAD

Flavin adenine dinucleotide

FCS

Fetal calf serum

GC-MS

Gas chromatography mass spectrometry

HRMS

High-resolution mass spectrometry

MAO

Monoamine oxidase

MS

Mass spectrometry

PBS

Phosphate-buffered saline

SDS-PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TAMRA

5(6)-carboxytetramethylrhodamine

TBTA

Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine

TCEP

Tris(2-carboxyethyl)phosphine

TLC

Thin-layer chromatography

THF

Tetrahydrofuran

Notes

Acknowledgements

The research by the authors was funded by the Austrian Science Fund (FWF) grant W901-B05 DK Molecular Enzymology and profited tremendously from the collaboration with Prof. S. A. Sieber (TU Munich, Germany), Prof. D. E. Edmondson (Emory University, Atlanta, USA) and Prof. Dr. W. Berger (Medical University of Vienna, Vienna).

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Chair of Organic Chemistry IITechnische Universität MünchenGarchingGermany
  2. 2.Institute of Organic ChemistryGraz University of TechnologyGrazAustria

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