Journal: Molecular & Cellular Proteomics : MCP
Article Title: A Method for Large-scale Identification of Protein Arginine Methylation *
Figure Lengend Snippet: A , Schematic representation of our workflow. Cells were labeled with light or heavy methionine, lysed and combined in a 1:1 protein ratio. Proteins were digested with trypsin or, in some experiments, with chymotrypsin. Arginine methylated peptides (depicted as red lines ) were enriched through the use of SCX, IEF (in an OFFGEL apparatus) or HILIC and analyzed by mass spectrometry. MS spectra for putative arginine methylated peptides were manually verified for the presence of a 1:1 methyl-SILAC pair. An example methyl-SILAC pair is shown. For each separation method, the total number of Meth-R sites identified as well as the novel ones is indicated; B , Meth-R-containing tryptic peptides have distinct physico-chemical properties. The Table indicates maximum, minimum and median values for isoelectric points (pI) and GRAVY scores of Meth-R peptides identified with the indicated enrichment methods. The number of unique peptides (n) from each method used to calculate values is shown. C , 3D scatter plot of 33000 non-methylated tryptic peptides ( gray circles ) identified from pH3–11 IPG strip and 128 arginine methylated tryptic peptides ( yellow-red circles ) identified from anti-DMA-IP, HILIC, SCX and IEF. Three different views of the same plot are shown. The peptides are plotted according to pI, GRAVY score and charge state at pH 2.7, representing the different selection criteria exploited by the enrichment methods. Yellow represents a low pI and red a high pI.
Article Snippet: Trypsin digests of Methyl-SILAC Jurkat or primary CD4+ lymphocytes were loaded onto a HILIC column in 90% acetonitrile.
Techniques: Labeling, Methylation, Electrofocusing, Hydrophilic Interaction Liquid Chromatography, Mass Spectrometry, Stripping Membranes, Selection