A method for large-scale identification of protein arginine methylation

Abstract

The lack of methods for proteome-scale detection of arginine methylation restricts our knowledge of its relevance in physiological and pathological processes. Here we show that most tryptic peptides containing methylated arginine(s) are highly basic and hydrophilic. Consequently, they could be considerably enriched from total cell extracts by simple protocols using either one of strong cation exchange chromatography, isoelectric focusing, or hydrophilic interaction liquid chromatography, the latter being by far the most effective of all. These methods, coupled with heavy methyl-stable isotope labeling by amino acids in cell culture and mass spectrometry, enabled in T cells the identification of 249 arginine methylation sites in 131 proteins, including 190 new sites and 93 proteins not previously known to be arginine methylated. By extending considerably the number of known arginine methylation sites, our data reveal a novel proline-rich consensus motif and identify for the first time arginine methylation in proteins involved in cytoskeleton rearrangement at the immunological synapse and in endosomal trafficking.

Fig. 1.

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.

Fig. 2.

A, Venn diagrams of the number of Meth-R sites identified by HILIC, IEF, and SCX individually and combined (left) or unique in HILIC or 7E6 mAb or found by both methods (right) in Jurkat cells and primary human CD4+ lymphocytes combined. B, Venn diagram showing the number of Meth-R sites found only in this study, the Meth-R sites annotated in UniProtKB database and those in common. C, Venn diagram of the number of arginine methylated proteins identified in this study, those previously reported in the UniProtKB database and those in common. Circles are approximately scaled relative to number of sites.

Fig. 3.

A, Sequence logo (41) representation of all arginine methylation sites identified in this study. Of methylated arginines, 57% were followed by a glycine and were generally flanked by a glycine rich sequence. The remaining 43% of sites reveal a previously unreported proline-rich arginine methylation (PRAM) motif. B, Frequency of proline at each position relative to the methylated arginine. Dashed lines indicate frequency level of proline with a probability of occurrence by chance of 0.05 or 0.01. p values for proline frequency at position –1 and +4 are shown. p values were calculated by bootstrapping. C, Most common motifs found at Meth-R sites. Note that motifs can be composed of combinations of motifs listed.

Fig. 4.

Breakdown of arginine methylated proteins according to distribution in cellular compartments (A) or by molecular function (B) according to Gene Ontology (GO) annotation.

Fig. 5.

Overview of identified proteins containing methylated arginine/lysine. Proteins are grouped according to annotated function in the UniprotKB database. Proteins newly identified as PRMT substrates in this study are drawn in dark blue. Previously known PRMT substrates are shaded in gray. Individual arginine methylation sites are marked as dots, blue representing novel Meth-R sites and gray representing previously known Meth-R sites. Green denotes a protein methylated on lysine.