To check whether the pronounced phosphotyrosine peptide recognition and discrimination prevailed in biological samples we decided to stepwise increase the protein sample Mass signals of all spiked peptides are found in the FT and W fractions whereas none in the E fraction. However, a different picture emerged from the spectra corresponding to the YpY-, pYY-and pYpY-MIP based enrichment fractions (Fig. ). In the enrichment using the YpY-MIP where YY, YpY, and pYpY peptides were spiked in the digest, the E fraction now revealed one single signal with a m/z matching the target YpY peptide (m/z = 1198.10) (Fig. ).