Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation

Abstract

Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine; asymmetric dimethyl arginine; and monomethyl, dimethyl, and trimethyl lysine motifs. These antibodies were used to perform immunoaffinity purification of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell line and mouse tissues, and ∼160 lysine methylation sites in human cell line HCT116. The number of methylation sites identified in this study exceeds those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared with those found in mouse embryo shows a tissue-specific distribution of arginine methylation, and extends the types of proteins that are known to be arginine methylated to include many new protein types. Many arginine-methylated proteins that we identified from the brain, including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, whereas the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.

Quantitative Profiling of Post-translational Modifications by Immunoaffinity Enrichment and LC-MS/MS in Cancer Serum without Immunodepletion

“…The top five protein categories for Rme were receptor/channel/transporter/cell surface protein, RNA processing, transcriptional regulator, adhesion/extracellular protein, and adaptor/scaffold. A large number of unique Rme sites was identified from various heterogeneous nuclear ribonucleoprotein (hnRNP) isoforms, most of which have been identified before in a previous study (14). The presence of these posttranslationally modified hnRNP peptides in serum/plasma has not been previously reported.…”

“…following protocols described previously (14,15). Briefly, saturating amounts of the indicated antibodies were bound to 30 l packed Protein A agarose beads (Roche, Indianapolis, IN) overnight at 4°C.…”

“…Sample Preparation-Serum samples were processed using the PTMScan method as previously described (14). Equal volumes of serum (250 l for individual sample) were mixed with urea lysis buffer (9 M sequanol-grade urea, 20 mM HEPES, pH 8.0, 1 mM ␤-glycerophosphate, 1 mM sodium vanadate, 2.5 mM sodium pyrophosphate) to a final concentration of 6 M urea.…”

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

“…Mass spectrometry-based proteomics is a well-established tool for the global identification of post-translational modification (PTM) sites and can be effectively coupled with immunoaffinity enrichment of peptides using "pan-specific" antibodies that bind to modified peptides independent of surrounding sequence context (19,20). Enrichment of modified peptides using panspecific antibodies has allowed for the identification of thousands of acetylated and ubiquitinated lysine sites in cells (21)(22)(23), and we and others have shown that pan-specific methyl-lysine or methyl-arginine antibodies can effectively and robustly enrich methylation modifications as well (7,8). Immunoaffinity enrichment is particularly critical in the case of methyl-lysine peptides, as the methyl moiety itself imparts sufficiently little physiochemical change to the side chain, rendering the enrichment of these peptides refractory to conventional separation methods.…”

“…Protein lysine methyltransferases (PKMTs) 1 catalyze the sequence-specific transfer of one, two, or three methyl groups to the side chains of lysine residues (1-4). In addition to the extensively studied lysine methylation on histones, PKMTs can modify non-histone proteins (3,(5)(6)(7)(8)). An increasing number of non-histone proteins have been reported as PKMT substrates, and, as a result, potential roles for the dysregulation of non-histone lysine methylation in cancer development and progression have been proposed (9).…”