The majority of eukaryotic proteins are phosphorylated in vivo, and phosphorylation may be the most common regulatory posttranslational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases and can be applied to studies of other protein modifications as well.
Bibliographical noteFunding Information:
This work was supported in part by National Science Foundation grant MCB 0841748 to M.C.H. and by the Purdue Center for Cancer Research Small Grants Program. This publication was also made possible by support from the Indiana Clinical and Translational Sciences Institute, funded in part by grant RR 02576 from the National Institutes of Health , National Center for Research Resources . J.S.M. was supported in part by a Purdue Research Foundation graduate assistantship.
- Enzymatic assay
- Label-free quantification
- Mass spectrometry
- Selected reaction monitoring