Stable isotope labeling with amino acids in cell culture (SILAC) has become an extremely valuable tool in quantitative proteomics and in biomarker discovery. Incorporation of SILAC labels occurs when cells are passaged multiple times in media where the endogenous amino acids are replaced with the heavy isotope ones. During a typical experiment, cells from heavy and light strains are combined in equal ratios and all steps of protein extraction and digestion occur on these cells together, minimizing the number of external variables introduced during sample processing. Potential biomarkers are revealed during liquid chromatography-tandem mass spectrometry analysis by peptides that differ considerably in intensity between the two strains as revealed by the mass shift from the incorporated SILAC label. The protocol presented here describes how to perform a typical experiment using the SILAC technology for the search for biomarkers as revealed by differences in protein expression levels, as well as by phosphorylation, a common posttranslational modification.
SILAC Biomarkers Phosphoproteomics Phosphopeptide FASP FACE TiO2IMAC
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