Notable features are the absolute discrimination of all phosphorylated forms of peptides featuring two juxtaposed tyrosines (Y492Y493), the high specificity and affinity for a doubly phosphorylated peptide and the high apparent recovery observed from native samples. This complements phosphospecific antibodies for this sequence motif that commonly exhibit undesirable crossreactivities. We foresee this kind of precision imprinting to be broadly applicable paving the way for robust, low-cost multiplex phosphopeptide assays compatible with clinical proteomics platforms. Binding Isotherms. The imprinted polymers (10 mg) were separately suspended in 1 mL solution of different concentrations (0 -100 µM) of the peptides (GADDSYYTAR, GADDSYpYTAR, GADDSpYYTAR, GADDSpYpYTAR) in MeCN/water: 90/10 (0.1% TFA) or in potassium phosphate (0.1M) buffer at pH = 7.4. The vials were shaken for 2 h followed by centrifugation and quantification of unbound analyte (Cfree) by HPLC using the method described above. The amount of bound analyte per unit mass of polymer (B) was calculated according to equation 1 from the total peptide concentration (C0), Cfree, volume (V) and polymer mass (m). The binding curves were then constructed by plotting B (µmol/g) against Cfree, and were subsequently fitted using the GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, USA) and the Langmuir mono-site binding model ( ):