title: Mass spectrometric real-time monitoring of an enzymatic phosphorylation assay using internal standards and data-handling freeware.
abstract: BACKGROUND
  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.
authors.affiliation: Zentrum für Forschung und Weiterbildung, Weihenstephan-Triesdorf - University of Applied Science, Am Staudengarten 7, D-85354, Freising-Weihenstephan, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Krappmann
authors.affiliation: Faculty of Sciences, Division of Chemistry, Department of Analytical Chemistry and Applied Spectroscopy, Free University of Amsterdam, De Boelelaan 1083, NL-1081 HV, Amsterdam, The Netherlands.
authors.forename: Arjen R
authors.initials: AR
authors.lastname: de Boer
authors.affiliation: Faculty of Sciences, Division of Chemistry, Department of Analytical Chemistry and Applied Spectroscopy, Free University of Amsterdam, De Boelelaan 1083, NL-1081 HV, Amsterdam, The Netherlands.
authors.forename: Daniël R W
authors.initials: DR
authors.lastname: Kool
authors.affiliation: Faculty of Sciences, Division of Chemistry, Department of Analytical Chemistry and Applied Spectroscopy, Free University of Amsterdam, De Boelelaan 1083, NL-1081 HV, Amsterdam, The Netherlands.
authors.forename: Hubertus
authors.initials: H
authors.lastname: Irth
authors.affiliation: Faculty of Sciences, Division of Chemistry, Department of Analytical Chemistry and Applied Spectroscopy, Free University of Amsterdam, De Boelelaan 1083, NL-1081 HV, Amsterdam, The Netherlands.|Analytical Research Group, Chair of Urban Water Systems Engineering, Technical University of Munich, Am Coulombwall 8, D-85748, Garching, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Letzel
mesh_terms: D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods* / Q000592:standards; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards; D008956:Models, Chemical; D009842:Oligopeptides / Q000032:analysis / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D012015:Reference Standards; D012815:Signal Processing, Computer-Assisted; D012984:Software
chemical_list: D009842:Oligopeptides; D010455:Peptides; C037642:kemptide; C064117:malantide; D017868:Cyclic AMP-Dependent Protein Kinases