16 Mass Spectrometry for Protein Quantification in Biomarker Discovery
205
Fig. 3. iTRAQ workflow and data analysis process. (a) iTRAQ labeling and MS data acquisition. (b) Protein identification is carried out based on the MS/MS fragmentation patterns, while relative quantification is determined by comparing the peak intensities of the iTRAQ reporter ions.
3. Centrifuge at 12,000 × g to pellet insoluble materials. 4. Determine the total protein concentration using the Bradford assay (34). 5. It is generally recommended to clean up samples by acetone precipitation (to remove any undesired interfering agents, e.g., DTT and detergents). Add 6 volumes of cold acetone to the cold sample tube, invert the tube three times, incubate at −20°C until a precipitate forms (~1 h). Centrifuge and decant the acetone. Air-dry. 6. To each tube containing 5–100 mg of sample, add 20 ml Dissolution Buffer.