Likewise, the pYY-MIP based enrichment, where YpY was spiked instead of pYY, led to a similar result (Fig. ). Targetting the doubly phosphorylated peptide, the pYpY-MIP was tested with the same spiked digest as used in YpY-MIP enrichment. Again, the target peptide was specifically enriched on this MIP as shown by the appearance of a single signal with a m/z matching pYpY (m/z = 1278.09) (Fig. ). Meanwhile YpY was detected in both the FT and W fractions. In all the aforementioned enrichments, the non-phosphorylated peptide YY (m/z= 1118.11) was mainly recovered in the FT fractions. Increasing sample complexity further, we tested a 12-protein digest in the same manner as above but with the model peptides spiked at the 1/10, 1/100, and 1/1000 levels. Fractions were collected from each step and analyzed by nanoliter flow LC-MS/MS (Orbitrap mass analyzer). Fig. (spiking level is 1:10) and Fig. (spiking level is 1:100) show the extracted ion chromatograms corresponding to the pre-enrichment control (reference, R) sample and the post-enrichment (flow through + wash, FT+W and elution, E) fractions.