Mass spectrometric real-time monitoring of an enzymatic phosphorylation assay using internal standards and data-handling freeware.

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.