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Overall Experimental Design. Cells are grown in D-MEM medium supplemented with either regular lysine, lysine-d4, and lysine-13C615N2 for 160hrs to allow complete mass-tagging of the corresponding proteome. The lysine-d4 and lysine-13C615N2 labeled macrophages were stimulated respectively by LPS for 20 hrs. Soluble protein from the three different cell populations are mixed equally and separated on 1D-SDS PAGE, followed by in-gel tryptic digestion and LC-MS analysis.
