Systematic development of an enzymatic phosphorylation assay compatible with mass spectrometric detection

A.V. den Boer, T. Letzel, H. Lingeman, H. Irth

    Research output: Contribution to JournalArticleAcademicpeer-review

    Abstract

    The enzymatic peptide phosphorylation by cAMP-dependent protein kinase A (PKA) was optimized and monitored by means of electrospray ionization mass spectrometry (ESI-MS). The direct detection of phosphorylated peptides by MS renders labeling unnecessary, reduces time and labor, due to less initial sample pretreatment. In this study the phosphorylation of the peptide malantide by PKA was performed in batch and reaction compounds were detected by ESI-MS after the incubation time. The subsequent product quantitation was accomplished by using one-point normalization. Applying this set-up, optimum solvent conditions (such as salt and modifier content), concentrations of essential reaction compounds (such as cAMP, Mg
    Original languageEnglish
    Pages (from-to)647-55
    JournalAnalytical and Bioanalytical Chemistry
    Volume381
    Issue number3
    DOIs
    Publication statusPublished - 2005

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