Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation

Cell Signaling Technology, United States
Molecular &amp Cellular Proteomics (Impact Factor: 7.25). 10/2013; 13(1). DOI: 10.1074/mcp.O113.027870
Source: PubMed

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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Recent evidence points to the protein arginine methyltransferase (PRMT) family of enzymes playing critical roles in cancer. PRMT7 has been identified in several gene expression studies to be associated with increased metastasis and decreased survival in breast cancer patients. However, this has not been extensively studied. Here we report that PRMT7 expression is significantly upregulated in both primary breast tumour tissues and in breast cancer lymph node metastases. We have demonstrated that reducing PRMT7 levels in invasive breast cancer cells using RNA interference significantly decreased cell invasion in vitro and metastasis in vivo. Conversely, overexpression of PRMT7 in non-aggressive MCF7 cells enhanced their invasiveness. Furthermore, we show that PRMT7 induces the expression of matrix metalloproteinase 9 (MMP9), a well-known mediator of breast cancer metastasis. Importantly, we significantly rescued invasion of aggressive breast cancer cells depleted of PRMT7 by the exogenous expression of MMP9. Our results demonstrate that upregulation of PRMT7 in breast cancer may have a significant role in promoting cell invasion through the regulation of MMP9. This identifies PRMT7 as a novel and potentially significant biomarker and therapeutic target for breast cancer.
    Oncotarget 12/2014; · 6.63 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 03/2015; DOI:10.1074/jbc.M115.641530 · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Arginine methylation is a novel post-translational modification within the voltage-gated ion channel superfamily, including the cardiac sodium channel, NaV1.5. We show that NaV1.5 R513 methylation decreases S516 phosphorylation rate by 4 orders of magnitude, the first evidence of protein kinase A inhibition by arginine methylation. Reciprocally, S516 phosphorylation blocks R513 methylation. NaV1.5 p.G514C, associated to cardiac conduction disease, abrogates R513 methylation, while leaving S516 phosphorylation rate unchanged. This is the first report of methylation-phosphorylation cross-talk of a cardiac ion channel.
    Amino Acids 12/2014; 47(2). DOI:10.1007/s00726-014-1890-0 · 3.65 Impact Factor