Absolute quantification of AMPK α-, β- and γ-subunit expression across human, mouse and rat tissues is notably lacking in the literature. This information would allow tissue AMPK activites to be quantified more accurately and give an indication of heterotrimer stoichiometries in rodent versus human tissues, relevant for studies on rodent disease models and on human diseases. The data would be important for choosing suitable rodent tissue and cell models for drug targeting specific AMPK complexes to treat human diseases.
We have developed a method for the absolute quantification of AMPK α-, β- and γ-subunits by LC-MS/MS using stable isotope-labeled internal standards1. This method is based on a QconCAT (Quantification conCATamer) reference construct corresponding to an artificial protein generated by concatenation of proteotypic and quantotypic peptides. We have selected a set of unique peptides to detect and quantify each of the AMPK subunits. Several methods for AMPK enrichment have been tested on mouse liver, white adipose tissue, heart and skeletal muscle. Immunoprecipitation2 was found to give the best results coherent with tissue AMPK subunit immunoblotting patterns described in the literature. Once the method has been validated, a comprehensive study of AMPK subunit quantification in rat, mouse along with human biobank tissues will be undertaken. Perspectives include studying tissue AMPK subunit levels in human cancer, obesity and diabetes. The methodology would also allow quantification of AMPK subunits in tissue/cell organelles after subcellular fractionation. Implications for the control of white adipose tissue metabolism by AMPK will be discussed.