Volume 18, Issue 8 p. 739-754
Full Paper

Characterisation of Photoaffinity-Based Chemical Probes by Fluorescence Imaging and Native-State Mass Spectrometry

Kanae Teruya

Kanae Teruya

Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Queensland, 4111 Australia

School of Natural Sciences, Griffith University, Nathan, Queensland, 4111 Australia

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Dr. Gregory M. Rankin

Dr. Gregory M. Rankin

Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Queensland, 4111 Australia

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Dr. Panagiotis K. Chrysanthopoulos

Dr. Panagiotis K. Chrysanthopoulos

Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Queensland, 4111 Australia

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Prof. Kathryn F. Tonissen

Corresponding Author

Prof. Kathryn F. Tonissen

Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Queensland, 4111 Australia

School of Natural Sciences, Griffith University, Nathan, Queensland, 4111 Australia

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Prof. Sally-Ann Poulsen

Corresponding Author

Prof. Sally-Ann Poulsen

Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Queensland, 4111 Australia

School of Natural Sciences, Griffith University, Nathan, Queensland, 4111 Australia

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First published: 08 February 2017
Citations: 6

Graphical Abstract

Chemical probe structure–activity relationships: Chemical probes are small-molecule reagents used by researchers for labelling and detection of biomolecules. We demonstrate that a combination of in-gel fluorescence with native state mass spectrometry enables detailed characterisation of photoaffinity labelling (PAL) probe–protein interactions to establish probe structure–activity relationships.

Abstract

Chemical probes are small-molecule reagents used by researchers for labelling and detection of biomolecules. We present the design, synthesis, and characterisation of a panel of 11 structurally diverse photoaffinity labelling (PAL) probes as research tools for labelling the model enzyme carbonic anhydrase (CA) in challenging environments, including in protein mixtures and cell lysates. We targeted the ubiquitous CA II as well as the two cancer-associated CAs (CA IX and CA XII) that are of high priority as potential biomarkers of aggressive and/or multidrug-resistant cancer. We utilise an atypical biophysical approach, native state mass spectrometry, to monitor the initial protein–probe binding and subsequent UV crosslinking efficiency of the protein:probe complex. This mass spectrometry methodology represents a new approach for chemical probe optimisation and development that might have broader applications to chemical probe characterisation beyond this study. This also represents one of the first studies, to the best of our knowledge, in which a comprehensive set of PAL probes has been used to establish the relationship between probe structure, noncovalent protein–probe binding, and covalent protein–probe crosslinking efficiency. Our results demonstrate the benefits of a comprehensive analysis of chemical probe structure–activity relationships to support the development of optimum chemical probes.

Conflict of interest

The authors declare no conflict of interest.