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. Author manuscript; available in PMC: 2018 Oct 3.
Published in final edited form as: Methods Mol Biol. 2016;1355:53–70. doi: 10.1007/978-1-4939-3049-4_4

Fig. 2.

Fig. 2

Experimental workflow for iTRAQ-based quantitative phosphoproteomic analysis. Cultured cells/tissue suspensions are treated with or without hormone for the indicated times followed by lysis in 8 M urea. Protein lysates are then digested with trypsin, desalted, and labeled with 8plex iTRAQ reagents. Strong cation exchange chromatography (SCX) stratifies the sample into 20 fractions followed by either immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC), which will enrich each fraction for phosphopeptides. Phosphopeptides are analyzed by tandem mass spectrometry (LC-MS/MS) in which the fragmentation is performed by higher energy collision induced dissociation (HCD), and the mass-to-charge ratio (m/z) and intensity of corresponding peptide ions are measured by an orbitrap-based mass spectrometer. In the MS2 spectrum, the pattern of b and y ions allows for phosphopeptide identification through database searching (black peaks), while the intensities of the iTRAQ reporter ions allow for relative quantification of phosphopeptide abundances across the eight different experimental conditions (colored peaks)