Characterisation of Photoaffinity-Based Chemical Probes by Fluorescence Imaging and Native-State Mass Spectrometry.
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.
Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Queensland, 4111, Australia.|School of Natural Sciences, Griffith University, Nathan, Queensland, 4111, Australia.
Kanae
K
Teruya
Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Queensland, 4111, Australia.
Gregory M
GM
Rankin
Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Queensland, 4111, Australia.
Panagiotis K
PK
Chrysanthopoulos
Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan, Queensland, 4111, Australia.|School of Natural Sciences, Griffith University, Nathan, Queensland, 4111, Australia.
Kathryn F
0000-0002-1018-2798
KF
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.
Sally-Ann
0000-0003-4494-3687
SA
Poulsen
D000818:Animals; D001577:Benzophenones / Q000138:chemical synthesis / Q000737:chemistry; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D045744:Cell Line, Tumor; D006801:Humans; D057054:Molecular Imaging; D061848:Optical Imaging; D010047:Ovalbumin / Q000737:chemistry; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000138:chemical synthesis / Q000737:chemistry
D001577:Benzophenones; D019920:Photoaffinity Labels; D013449:Sulfonamides; D012710:Serum Albumin, Bovine; D010047:Ovalbumin; D002256:Carbonic Anhydrases
benzophenone; click chemistry; in-gel fluorescence; mass spectrometry; photoaffinity labeling