Figure 1

Monitoring changes in protein phosphorylation by the SILAC method. (a) Schematic outline of the method. Separate cultures of cells are grown in normal medium (¹²C6-arginine) or in medium containing arginine labeled at all six carbons with ¹³C (¹³C6-arginine). The cells in normal medium are left unstimulated whereas cells in the ¹³C-arginine medium are stimulated with an agent that activates signaling. The cells are harvested and equal amounts of lysate protein mixed together. In most cases, steps to enrich phosphoproteins, enrich phosphopeptides after trypsin digestion, or both, are needed to detect low-abundance phosphopeptides. The peptides are resolved by reverse-phase liquid chromatography (LC) followed by online mass spectrometry (MS). Tandem mass spectrometry (MS/MS) data are used for automated database searching to identify peptides (and their corresponding protein) and to detect phosphopeptides (typically by detecting neutral loss of phosphate during MS/MS). In many cases, the MS/MS data can also be used to assign site(s) of phosphorylation. Once peptides of interest are identified, the relative amounts of peptide derived from the unstimulated cells (grown in normal medium) and the stimulated cells (grown in ¹³C-arginine medium) are determined from high-resolution MS scans. (b, c) Quantitation of peptides. (b) A total ion chromatogram of a protein digest eluted from a reverse-phase column and analyzed by mass spectrometry. The peaks represents the total ion signal from individual peptides. (c) shows high-resolution MS scans of a non-phosphorylated peptide (left panel) and a different phosphorylated peptide (right panel), which was identified from MS/MS scans (not shown), eluted from the reverse-phase column at different retention times. The left-hand panel is a magnified MS scan showing the normal and ¹³C-arginine-labeled versions of the non-phosphorylated peptide with mass-to-charge ratio (m/z) of 588. Even though the ¹³C-arginine peptide has a mass that is 6 Da higher than the normal peptide, m/z differs by only 3 (m/z 591) because the peptide has two positive charges (2⁺). Both peptides appear as a series of isotopic peaks as a result of the natural abundance of heavy isotopes. The relative amounts of the normal and ¹³C-arginine peptides are determined by comparing the area under the monoisotopic peaks (the tallest peak in each series) of each peptide. In this example experiment, the amount of this non-phosphorylated peptide was not affected by stimulation and the ratio of the two peptides is 1.0. The right-hand panel shows the same data for the phosphopeptide of m/z 628 that eluted at 35 minutes. In this case, the ratio of the normal peptide to the ¹³C-arginine peptide is 0.2, showing that the amount of phosphopeptide increased fivefold following stimulation.