PRM assays are first characterized by performing a response curve assay to identify the lower limit of quantification (LLOQ), limit of detection (LOD) and linear range for each surrogate peptide. Briefly, a  background matrix consisting on a MCF7 cell lysate was digested with trypsin. Reverse curves were prepared in triplicate by varying the concentration of the heay-labelled peptide standards over nine concentration points (1000, 200, 66.6, 22.2, 7.4, 2.46, 0.82, 0.27, 0.09 fmol/μg). Light peptide was added at a constant concentration of 100 fmol/μg.

Surrogate peptides unique to the target proteins of interest (ER, TET2, GATA3 and Actin) were chosen and stable-isotope-labelled versions (SIS) of these peptides were synthesized.

A background matrix consisting on a MCF7 cell lysate was digested with trypsin. The resulting peptide mixture is then spiked with increasing concentrations of the heavy-labelled peptide standards and a constant concentration of the light version of the same surrogate peptides.

Curve blanks contained no heavy-labelled peptides.