Targeted mass spectrometry isotope analysis (i.e. selected reaction monitoring or SRM) is an alternative to antibody-based assays for the validation of clinically relevant biomarkers, but also has been employed for discovery-based quantitative proteomics (see example references below). One obstacle of this strategy is that every peptide possesses unique biochemical characteristics. Its amino acid composition and possible post-translational modifications defines its elution profile from the liquid chromatography column, ionization and fragmentation. For developing a diagnostic clinical MS assay, these peptide properties must be characterized with a synthesized peptide before analyzing the peptide of interest in vivo. Peptides also can be synthesized with heavy stable isotopes for absolute quantitation by spiking in a known amount of the heavy peptide into the biological sample. These strategies are also commonly employed to validate results from large scale quantitative proteomic analyses.