Figure 2: MARQUIS method for absolute quantification.

Synthetic isotope-labelled phosphorylated peptides (represented by the square box) are added during cell lysis. Following sample processing and digestion, all peptides, synthetic and endogenous (represented by the circle), are iTRAQ labelled. For this particular application of the method, tyrosine-phosphorylated peptides are enriched by peptide immunoprecipitation followed by immobilized metal affinity chromatography (IMAC), and analysed by LC–MS/MS. Heavy-labelled standards provide an internal calibration curve, enabling absolute quantification of endogenous peptides.