Protein phosphorylation stoichiometry was assessed bytwo analytical strategies. Both are based on element massspectrometry (ICPMS, inductively coupled plasma massspectrometry)
and simultaneous monitoring of
31P
and34S. One strategy employs a combination of 1D gelelectrophoresis, in-gel digestion,
and final
LC-ICPMSanalysis (
LC = capillary liquid chromatography). Theother strategy uses the combination of 1D gel electrophoresis, protein blotting,
and imLA-ICPMS (imLA =imaging laser ablation). The two methods were evaluatedwith st
andard phosphoproteins
and were applied to theanalysis of the cytoplasmatic proteome of bacterial cells(
Corynebacterium glutamicum)
and eukaryotic cells(
Mus musculus). The eukaryotic proteome was found toexhibit a significantly higher phosphorylation degree(~0.8 mol of P/mol of protein) compared to the bacterialproteome (~0.01 mol of P/mol of protein). Both analyticalstrategies revealed consistent quantitative results, with the
LC-ICPMS approach providing the higher sensitivity.In summary, two ICPMS-based methods for quantitativeestimation of the phosphorylation degree of a cellularproteome are presented which access the native proteomestate
and do not require any type of label introduction orderivatization.