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 respectively. These antibodies were used to perform immunoaffinity purification (IAP) 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 lines and mouse tissues, and approximately 160 lysine methylation sites in human cell line HCT116. The methylation sites that were identified in this study exceed those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared to those found in mouse embryo shows tissue-specific distribution of arginine methylation, and extends the type of proteins that are known to be arginine methylated to 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, while the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.
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ABSTRACT: The lysine methyltransferase (KMT) SETMAR is implicated in response to and repair of DNA damage but its molecular function is not clear. More broadly, enzyme/substrate relationships are largely unknown for the dozens of predicted KMTs and hundreds or thousands of proteins modified by lysine methylation. SETMAR has been associated with di-methylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates we utilized a strategy based on quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 (K130) of mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates mono-methylation at this position. Further, we show that SETMAR methylates snRNP70 K130 in cells. As snRNP70 is a key early regulator of 5' splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 05/2015; 290:12040. DOI:10.1074/jbc.M115.641530 · 4.60 Impact Factor
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ABSTRACT: The human genome encodes a family of nine protein arginine methyltransferases (PRMT1-9), whose members can catalyse three distinct types of methylation on arginine residues. Here we identify two spliceosome-associated proteins-SAP145 and SAP49-as PRMT9-binding partners, linking PRMT9 to U2 snRNP maturation. We show that SAP145 is methylated by PRMT9 at arginine 508, which takes the form of monomethylated arginine (MMA) and symmetrically dimethylated arginine (SDMA). PRMT9 thus joins PRMT5 as the only mammalian enzymes capable of depositing the SDMA mark. Methylation of SAP145 on Arg 508 generates a binding site for the Tudor domain of the Survival of Motor Neuron (SMN) protein, and RNA-seq analysis reveals gross splicing changes when PRMT9 levels are attenuated. These results identify PRMT9 as a nonhistone methyltransferase that primes the U2 snRNP for interaction with SMN.Nature Communications 03/2015; 6:6428. DOI:10.1038/ncomms7428 · 10.74 Impact Factor
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ABSTRACT: Protein methylation and acetylation play an important role in biological processes, and misregulation of these modifications are involved in various diseases. Therefore, it is critical to understand the activities of the enzymes responsible for these modifications. Herein we describe a sensitive method for ratiometric quantification of methylated and acetylated peptides via MALDI-MS by direct spotting of enzymatic protein methylation and acetylation reaction mixtures without tedious purification procedures. The quantifiable detection limit for peptides with our method is approximately 10 femtomoles. This is achieved by increasing the signal-to-noise ratio through addition of NH4H2PO4 to the matrix solution and reduction of the matrix CHCA concentration to 2 mg/mL. We have demonstrated the application of this method in enzyme kinetic analysis and inhibition studies. The unique feature of this method is the simultaneous quantification of multiple peptide species for investigation of processivity mechanisms. Its wide buffer compatibility makes it possible to be adapted for the investigation of the activity of any protein methyltransferases and acetyltransferases. Copyright © 2015 Elsevier Inc. All rights reserved.Analytical Biochemistry 03/2015; 478:59-64. DOI:10.1016/j.ab.2015.03.007 · 2.31 Impact Factor