Figure 2

Principles of quantitative proteomics. A) Label-free quantitation performed by peptide peak area under the curve. Proteins are extracted from tissue, proteolytically digested into peptides and analyzed by liquid chromatography (LC)-MS. Analyte intensity versus retention time profiles are generated from which area under the curve (AUC) or summed peak intensities are calculated. Relative peptide amount in healthy versus disease sample is proportional to peak AUC or summed intensities. Targeted peptide identification is typically performed on a subsequent injection. B) Label-based quantitation with the iTRAQ® (isotope tagging for relative and absolute quantitation) 4plex workflow. Proteins from four individual samples are digested into peptides that are tagged with isobaric stable isotope labeled chemicals. Four chemical tags have 4 unique mass-to-charge (m/z) values that are produced during peptide tandem MS (MS/MS) and used for relative quantitation by relative peak intensity. Peptide fragment ions are used for peptide ID and protein inference.