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Biochemistry

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Published: April 28th, 2022

DOI:

10.3791/62409

1Department of Experimental Oncology, European Institute of Oncology IRCCS (IEO), 2European School of Molecular Medicine (SEMM), c/o Campus IFOM-IEO
* These authors contributed equally

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.

In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.

Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Arginine (R)-methylation is a post translational modification (PTM) that decorates around 1% of the mammalian proteome1. Protein Arginine Methyltransferases (PRMTs) are the enzymes catalyzing R-methylation reaction by the deposition of one or two methyl groups to the nitrogen (N) atoms of the guanidino group of the side chain of R in a symmetric or asymmetric manner. In mammals, PRMTs can be grouped into three classes-type I, type II, and type III-depending on their capability to deposit both mono-methylation (MMA) and asymmetric di-methylation (ADMA), MMA and symmetric di-methylation (SDMA) or only MMA, respectively2

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1. Cell culturing and protein extraction (time: 3 - 4 weeks required)

  1. Grow HeLa cells in parallel in media supplied with either Light (L) or Heavy (H) Methionine, respectively (see Table 1 for media composition). Upon at least eight cell divisions, collect an aliquot of cells from each SILAC channel and perform the incorporation test.
    NOTE: To check for the incorporation efficiency, test by LC-MS/MS analysis that the percentage of heavy Methionine (Met-4) in the Heavy channel is as nea.......

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The article describes a workflow for the high-confidence identification of global protein R-methylation, which is based on the combination of the enzymatic digestion of the protein extract with two distinct proteases in parallel, followed by HpH-RP liquid chromatography fractionation of proteolytic peptides and immuno-affinity enrichment of R-methyl-peptides with anti-pan-R-methyl antibodies (Figure 1).

The cells were grown in the presence of Methionine, either na.......

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The high confidence identification of in vivo protein/peptide methylation by global MS-based proteomics is challenging, due to the risk of high FDR, with several amino acid substitutions and methyl-esterification occurring during sample preparation that are isobaric to methylation and can cause wrong assignments in the absence of orthogonal MS validation strategies. The substoichiometric nature of this PTM further complicates the task of global methyl-proteomics, but can be overcome with the selective enrichment.......

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MM and EM are PhD students within the European School of Molecular Medicine (SEMM). EM is the recipient of a 3-years FIRC-AIRC bursary (Project Code: 22506). Global analyses of R-methyl-proteomes in the TB group are supported by the AIRC IG Grant (Project Code: 21834).

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Name Company Catalog Number Comments
Ammonium Bicarbonate (AMBIC) Sigma-Aldrich 09830
Ammonium Persulfate (APS) Sigma-Aldrich 497363
C18 Sep-Pak columns vacc 6cc (1g) Waters WAT036905
Colloidal Coomassie staining Instant Sigma-Aldrich ISB1L-1L
cOmplete Mini, EDTA-free Roche-Sigma Aldrich 11836170001 Protease Inhibitor
Dialyzed Fetal Bovine Serum (FBS) GIBCO ThermoFisher 26400-044
DL-Dithiothreitol (DTT) Sigma-Aldrich 3483-12-3
DMEM Medium GIBCO ThermoFisher requested  with stabile glutamine and without methionine
EASY-nano LC 1200 chromatography system ThermoFisher
EASY-Spray HPLC Columns ThermoFisher ES907
Glycerolo Sigma-Aldrich G5516
HeLa cells ATCC ATCC CCL-2
HEPES Sigma-Aldrich H3375
Iodoacetamide (IAA) Sigma-Aldrich 144-48-9
Jupiter C12-RP column Phenomenex 00G-4396-E0
L-Methionine Sigma-Aldrich M5308 Light (L) Methionine
L-Methionine-(methyl-13C,d3) Sigma-Aldrich 299154 Heavy (H) Methionine
LysargiNase Merck Millipore EMS0008
Microtip Cell Disruptor Sonifier 250 Branson
N,N,N′,N′-Tetramethylethylenediamine (TEMED) Sigma-Aldrich T9281
Penicillin-Streptomycin GIBCO ThermoFisher 15140122
PhosSTOP Roche-Sigma Aldrich 4906837001 Phosphatase Inhibitor
Pierce C18 Tips ThermoFisher 87782
Pierce  0.1% Formic Acid (v/v) in Acetonitrile, LC-MS Grade ThermoFisher 85175 LC-MS Solvent B
Pierce  0.1% Formic Acid (v/v) in Water, LC-MS Grade ThermoFisher 85170 LC-MS Solvent A
Pierce  Acetonitrile (ACN), LC-MS Grade ThermoFisher 51101
Pierce  Water, LC-MS Grade ThermoFisher 51140
Polyacrylamide Sigma-Aldrich 92560
Precision Plus Protein  All Blue Prestained Protein Standards Bio-Rad 1610373
PTMScan antibodies α-ADMA Cell Signaling Technology 13474
PTMScan antibodies α-MMA Cell Signaling Technology 12235
PTMScan antibodies α-SDMA Cell Signaling Technology 13563
Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer ThermoFisher
Sequencing Grade Modified Trypsin Promega V5113
Trifluoroacetic acid Sigma-Aldrich T6508
Ultimate 3000 HPLC Dionex
Urea Sigma-Aldrich U5378
Vacuum Concentrator 5301 Eppendorf Speed vac

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