Because protein phosphorylation is a key means of initiating and propagating changes in cellular states, techniques to readily analyze and quantify the dynamics of protein phosphorylation can aid the study of cellular function. One such technique is stable isotope labeling with mass spectrometric readout.

For analysis of proteins, metabolic stable isotope labeling has the advantage over other isotope-incorporation techniques since label is introduced during cell growth and therefore, the labeled and unlabeled samples are mixed prior to cell lysis, ensuring that no artifactual differences are introduced during sample processing. Furthermore, metabolic labeling requires no derivatization steps that may lead to sample handling-related losses. However, growth of cells in labeled-medium to study proteins expressed at endogenous cellular levels, can be prohibitively expensive due to the cost of labeled medium.

We introduce an economically viable strategy using metabolic labeling for quantifying changes in phosphorylation at a two order-of-magnitude reduction in cost relative to typical metabolic labeling strategies. In our overexpression isotope-tag doping strategy, the phosphoprotein is purified from unlabeled cells under all conditions of interest. We then introduce a stable isotope labeled internal standard by doping them prior to cell lysis with small amounts of cells that overexpressed the same phosphoprotein while growing in stable isotope-labeled medium. After proteolytic cleavage, the labeled phosphopeptides from the overexpressing cells can be used for comparing phosphopeptide levels across all the samples of interest.