Figure 1.

Flow diagram of the SILAC MS/MS experiment. The UM-SCC-1 control and Pre375 cells were grown in medium supplemented with heavy (¹³C₆-lysine and ¹³C₆, ¹⁵N₄-arginine) or light (¹²C₆-lysine and ¹²C₆, ¹⁴N₄-arginine) amino acids, respectively. Equal amounts of heavy grown control cells and light grown Pre375 cells were combined and subcellular fractionation was conducted. The fractions were run on SDS-PAGE. The gel pieces were excised and subjected to in-gel tryptic digestion. The tryptic peptides from digested bands were analyzed by mass spectrometry. Alternating MS (left) and tandem MS (MS/MS; right) provided the intensity peak ratios between heavy- and light-labeled peptides and the identity of peptides acquired (data for a peptide from L-plastin are shown). LC, liquid chromatography; MS, mass spectrometry; RP, reverse phase.