Fig. 1

Experimental workflow for protein half-life determination using dynamic SILAC and quantitative mass spectrometry. Five non-dividing primary cell types comprising B-cells, NK cells, monocytes, hepatocytes, and mouse embryonic neurons were adapted to light SILAC medium. To label newly synthesized proteins, the cells were exposed to heavy SILAC medium and collected at different time points. After protein extraction, proteolysis with trypsin, sample preparation, and subsequent LC-MS/MS analysis, the peptides were identified by the Mascot database search engine and quantified using the isobarQuant software package. Peptides of pre-existing and newly synthesized proteins were distinguished by their mass due to incorporation of light or heavy arginine and lysine. Protein fold changes at different time points were calculated using the intensity ratios of heavy vs. light SILAC peptides and were used for subsequent protein half-life determination