Simultaneous analysis of enzyme structure and activity by kinetic capillary electrophoresis-MS.
To enable the detection of protein conformational isomers, their enzymatic activity and their inhibition in a single experiment, we developed a method based on kinetic capillary electrophoresis coupled on-line with UV detection and ion mobility mass spectrometry (CE-UV-IM-MS). Kinetic CE-UV separated protein conformers and monitored their interconversion dynamics in solution. Ion mobility mass spectrometry analyzed the conformer sizes, exact molecular weights, and structures of an enzyme and of its substrates, inhibitors and corresponding products. This coupled CE-UV-IM-MS system allowed the simultaneous, real-time observation of the effect of small-molecule inhibitors on both the conformational distribution and enzymatic activity of the human tissue transglutaminase TG2. By expanding mass spectrometry profiling of enzymatic reactions beyond proteins and substrates to include protein dynamics, CE-UV-IM-MS opens a new avenue for the modulation and regulation of cellular functions, drug development and protein engineering.
Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada.
Gleb G
GG
Mironov
Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada.
Christopher M
CM
Clouthier
Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada.
Abdullah
A
Akbar
Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada.
Jeffrey W
http://orcid.org/0000-0002-8133-6862
JW
Keillor
Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada.
Maxim V
http://orcid.org/0000-0003-0514-599X
MV
Berezovski
D004305:Dose-Response Relationship, Drug; D019075:Electrophoresis, Capillary; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D011503:Transglutaminases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins