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Document type:
Zeitschriftenaufsatz 
Author(s):
Krappmann, Michael; de Boer, Arjen R.; Kool, Daniël R.W.; Irth, Hubertus; Letzel, Thomas 
Title:
Mass spectrometric real-time monitoring of an enzymatic phosphorylation assay using internal standards and data-handling freeware 
Abstract:
Rationale Continuous-flow reaction detection systems (monitoring enzymatic reactions with mass spectrometry (MS)) lack quantitative values so far. Therefore, two independent internal standards (IS) are implemented in a way that the online system stability can be observed, quantitative conversion values for substrate and product can be obtained and they can be used as mass calibration standards for high MS accuracy. Methods An application previously developed for the MS detection of peptide phosphorylation by cAMP-dependent protein kinase A (PKA) (De Boer et al., Anal. Bioanal. Chem. 2005, 381, 647–655) was transferred to a continuous-flow reaction detection system. This enzymatic reaction, involving enzyme activation as well as the transfer of a phosphate group from ATP to a peptide substrate, was used to prove the compatibility of a quantitative enzymatic assay in a continuous-flow real-time system (connected to MS). Results Moreover (using internal standards), the critical parameter reaction temperature (including solution density variations depending on temperature) was studied in the continuous-flow mixing system. Furthermore, two substrates (malantide and kemptide), two enzyme types (catalytic subunit of PKA and complete PKA) and one inhibitor were tested to determine system robustness and long-term availability. Even spraying solutions that contained significant amount of MS contaminants (e.g. the polluted catalytic subunit) resulted in quantifiable MS signal intensities. Subsequent recalculations using the internal standards led to results representing the power of this application. Conclusions The presented methodology and the data evaluation with available Achroma freeware enable the direct coupling of biochemical assays with quantitative MS detection. Monitoring changes such as temperature, reaction time, inhibition, or compound concentrations can be observed quantitatively and thus enzymatic activity can be calculated. Copyright © 2016 John Wiley & Sons, Ltd. 
Keywords:
AF 
Journal title:
Rapid Communications in Mass Spectrometry 
Year:
2016 
Journal volume:
30 
Journal issue:
Pages contribution:
1019--1030 
Fulltext / DOI:
Print-ISSN:
1097-0231 
Notes:
RCM-15-0342.R2