Online quench-flow electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for elucidating kinetic and chemical enzymatic reaction mechanisms

Analytical Chemistry
David J ClarkeC Logan Mackay

Abstract

We have developed an automated quench-flow microreactor which interfaces directly to an electrospray ionization (ESI) mass spectrometer. We have used this device in conjunction with ESI Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to demonstrate the potential of this approach for studying the mechanistic details of enzyme reactions. For the model system chosen to test this device, namely, the pre-steady-state hydrolysis of p-nitrophenyl acetate by the enzyme chymotrypsin, the kinetic parameters obtained are in good agreement with those in the literature. To our knowledge, this is the first reported use of online quench-flow coupled with FTICR MS. Furthermore, we have exploited the power of FTICR MS to interrogate the quenched covalently bound enzyme intermediate using top-down fragmentation. The accurate mass capabilities of FTICR MS permitted the nature of the intermediate to be assigned with high confidence. Electron capture dissociation (ECD) fragmentation allowed us to locate the intermediate to a five amino acid section of the protein--which includes the known catalytic residue, Ser(195). This experimental approach, which uniquely can provide both kinetic and chemical details of enzyme mechanisms,...Continue Reading

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Citations

Sep 29, 2011·Journal of the American Society for Mass Spectrometry·David J ClarkePat R R Langridge-Smith
Dec 19, 2012·Journal of the American Society for Mass Spectrometry·Lu DengJohn S Klassen
Aug 13, 2013·Journal of the American Society for Mass Spectrometry·Jeremy S BarnesKevin A Schug
Dec 11, 2013·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Ryan MatsudaDavid S Hage
Sep 28, 2014·Free Radical Biology & Medicine·Nelmi O Devarie BaezCristina M Furdui
Feb 7, 2019·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Mumiye A OgunwaleXiao-An Fu
May 7, 2011·Chemical Society Reviews·Gili Ben-Nissan, Michal Sharon

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