Fig. 2.

Generation of a high quality reference MS/MS-spectral library. A, solubilized synthetic peptides are analyzed by electrospray ionization, with targeted isolation and fragmentation using an ion trap or triple quadrupole mass spectrometer and the product ions recorded continuously over 5 min. B, collected MS/MS spectra sequence-verified by database searching using SEQUEST. C, characteristic experimental intensity patterns of distinguishing b- and y-ion features stored in a relational database. D, MRM assay development using empirically optimized collision energy settings, shown here based on the breakdown curves obtained for the 10 most intense transitions (see “Experimental Procedures” and supplemental Methods” for details).