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Method Article
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, respectively2,3. PRMTs mainly target R residues located within glycine- and arginine-rich regions, known as GAR motifs, but some PRMTs, such as PRMT5 and CARM1, can methylate proline-glycine-methionine-rich (PGM) motifs4. R-methylation has emerged as a protein modulator of several biological processes, such as RNA splicing5, DNA repair6, miRNA biogenesis7, and translation2, fostering the research on this PTM.
Mass Spectrometry (MS) is recognized as the most effective technology to systematically study global R-methylation at protein-, peptide-, and site-resolution. However, this PTM requires some particular precautions for its high-confidence identification by MS. First, R-methylation is substoichiometric, with the unmodified form of the peptides being much more abundant than the modified ones, so that mass spectrometers operating in the Data Dependent Acquisition (DDA) mode will fragment high-intensity unmodified peptides more often than their lower-intensity methylated counterparts8. Moreover, most MS-based workflows for R-methylated site identification suffer from limitations at the bioinformatic analysis level. Indeed, the computational identification of methyl-peptides is prone to high False Discovery Rates (FDR), because this PTM is isobaric to various amino acid substitutions (e.g., glycine into alanine) and chemical modification, such as methyl-esterification of aspartate and glutamate9. Hence, methods based on the isotope labeling of methyl groups, such as Heavy Methyl Stable Isotope Labeling with Amino Acids in Cell culture (hmSILAC), have been implemented as orthogonal strategies for confident MS-identification of in vivo methylations, significantly reducing the rate of false positive annotations10.
Recently, various proteome-wide protocols to study R-methylated proteins have been optimized. The development of antibody-based strategies for the immuno-affinity enrichment of R-methyl-peptides has led to the annotation of several hundreds of R-methylated sites in human cells11,12. Furthermore, many studies3,13 reported that coupling antibody-based enrichment with peptide separation techniques such as HpH-RP chromatography fractionation can boost the overall number of methyl-peptides identified.
This article describes an experimental strategy designed for the systematic and high-confidence identification of R-methylated sites in human cells, based on various biochemical and analytical steps: protein extraction from hmSILAC-labeled cells, parallel double enzymatic digestion with Trypsin and LysargiNase proteases, followed by HpH-RP chromatographic fractionation of digested peptides, coupled with antibody-based immuno-affinity enrichment of MMA-, SDMA-, and ADMA-containing peptides. All affinity-enriched peptides are then analyzed by high-resolution Liquid Chromatography (LC)-MS/MS in DDA mode, and raw MS data are processed by MaxQuant algorithm for identificationof R-methyl-peptides. Finally, the MaxQuant output results are processed with hmSEEKER, an in-house developed bioinformatics tool to search pairs of heavy and light methyl-peptides. Briefly, hmSEEKER reads and filters methyl-peptides identifications from the msms file, then matches each methyl-peptide to its corresponding MS1 peak in the allPeptides file, and, finally, searches the peak of the heavy/light peptide counterpart. For each putative heavy-light pair, the Log2 H/L ratio (LogRatio), Retention Time difference (dRT), and Mass Error (ME) parameters are calculated, and doublets that are located within user-defined cut-offs are labeled as true positives. The workflow of the biochemical protocol is described in Figure 1.
1. Cell culturing and protein extraction (time: 3 - 4 weeks required)
2. Lysate digestion (indicative time required 2 hours)
3. Peptide purification (indicative time required 1 hour)
4. Coomassie-stained SDS-PAGE gel (indicative time required 2 hour)
5. Peptide lyophilization (indicative time 2 days)
6. Off-line HpH-RP chromatographic fractionation of peptides (indicative time 4 days)
7. R-methylated peptide immuno-affinity enrichment (indicative time 2 days)
8. Desalting and concentration of affinity-enriched methyl-peptides by C18 microcolumns (indicative time required 30 minutes)
9. Second enzymatic digestion (indicative time required 3 hours)
10. Desalting peptides (indicative time required 30 minutes)
11. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (indicative time 5 days)
12. Running MaxQuant and hmSEEKER data analysis
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...
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...
The authors have nothing to disclose.
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).
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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