Dataset Information

A novel strategy of profiling protein methylation on lysine and arginine

ABSTRACT: Protein methylation is involved in different processes including gene expression regulation, epigenetics, and nucleotide metabolism. Identification of methylated proteins is difficult as methyl groups are small, and do not introduce significant changes in protein hydrophobicity or charge state. The most effective analytical method to date is relative enrichment by pan-specific antibodies and methylation specific binding domains. Here, we present a novel and unbiased chemical strategy to enrich and identify sites of lysine and arginine methylations. This approach makes use of the property that methylation does not alter the charge state of lysine and arginine. This approach revealed over 793 methylation events including 211 arginine and 585 lysine methylation sites in HEK 293 Cells. This strategy proves to be convenient, effective and versatile, with the potential of analyzing other PTMs on both lysine and arginine.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Zhibin Ning

LAB HEAD: Daniel Figeys

PROVIDER: PXD001689 | Pride | 2016-08-16

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Anna_20120622_Methylation_MDA_OPA_SCX_pH030.RAW	Raw
	Anna_20120622_Methylation_MDA_OPA_SCX_pH030.dat	Other
	Anna_20120622_Methylation_MDA_OPA_SCX_pH030_120625072926.RAW	Raw
	Anna_20120622_Methylation_MDA_OPA_SCX_pH030_120625072926.dat	Other
	Anna_20120622_Methylation_MDA_OPA_SCX_pH040.RAW	Raw

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Publications

A charge-suppressing strategy for probing protein methylation.

Ning Zhibin Z Star Alexandra Therese AT Mierzwa Anna A Lanouette Sylvain S Mayne Janice J Couture Jean-Francois JF Figeys Daniel D

Chemical communications (Cambridge, England) 20160401 31

Methylation of arginine and lysine (RK) residues play essential roles in epigenetics and the regulation of gene expression. However, research in this area is often hindered by the lack of effective tools for probing the protein methylation. Here, we present an antibody-free strategy to capture protein methylation on RK residues by using chemical reactions to eliminate the charges on un-modified RK residues and peptide N-termini. Peptides containing methylated RK residues remain positively charge ...[more]

PMID: 27021271

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Project description:Protein methylation has been implicated in many important biological contexts including signaling, metabolism, and transcriptional control. Despite the importance of this post- translational modification, the global analysis of protein methylation by mass spectrometry-based proteomics has not been extensively studied due to the lack of robust, well-characterized techniques for methyl-peptidemethyl peptide enrichment. Here, to better investigate protein methylation, we optimized and compared two methods for methyl-peptidemethyl peptide enrichment: immunoaffinity purification (IAP) and high pH strong cation exchange (SCX). Comparison of these methods revealed that they are largely orthogonal for monomethyl arginine (MMA), suggesting that the usage of both techniques is required to provide a global view of protein methylation. Using both IAP and SCX, we investigated changes in protein methylation downstream of protein arginine methyltransferase 1 (PRMT1). Together, these techniques allowed us to quantify over ~1,000 arginine methylation sites on 407 proteins. Of these methylation sites, PRMT1 knockdown resulted in significant changes to 110 arginine methylation sites on 59 proteins. In contrast, zero lysine methylation sites were significantly changed upon PRMT1 knockdown. In PRMT1 knockdown cells, 24 MMA sites exhibited both significant downregulation (ie, putative PRMT1-mediated MMA targets) and 63 significant upregulation (ie, putative PRMT1-mediated ADMA targets). PRMT1 knockdown induced significant changes in asymmetric dimethyl arginine (ADMA), consistent with being PRMT1 targets. Additionally, We also observed that PRMT1 knockdown induced significant increases in symmetric dimethyl arginine (SDMA) methylation, suggesting that loss of PRMT1 activity allows scavenging of PRMT1 substrates by Type II PRMTs. Analysis of the subcellular localization and protein function of the significantly changing methyl-proteins revealed that the majority of PRMT1 substrates are localized to the nucleus and involved in RNA and DNA binding. Taken together, our results suggest that deep protein methylation profiling by mass spectrometry requires orthogonal enrichment techniques, identify novel PRMT1 methylation targets, and highlight in order to understand the dynamic interplay between methyltransferases in mammalian cells.