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

Summary

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.

Abstract

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.

Introduction

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

Protocol

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 near as possible to 100%. Analyze an aliquot of heavy-labeled cells by LC-MS/MS (for settings see Table 2), then process the MS data wit....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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