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. Author manuscript; available in PMC: 2023 May 20.
Published in final edited form as: Methods Enzymol. 2019 Aug 1;626:89–113. doi: 10.1016/bs.mie.2019.07.028

Biochemical analysis of protein arginylation

Junling Wang a, John R Yates III b, Anna Kashina a,*
PMCID: PMC10199459  NIHMSID: NIHMS1897786  PMID: 31606094

Abstract

Protein arginylation—enzymatic addition of the amino acid arginine (Arg) to proteins, mediated by arginyltransferase ATE1, has been discovered in 1963, but is still relatively poorly understood. Studies of arginylation present many technical challenges, which arise from the fact that Arg is a regular amino acid that also incorporates into proteins during translation. Thus, in vitro arginylation needs to be conducted in a strictly ribosome-free system, in highly controlled conditions. Identification of arginylated proteins is currently only possible by high precision mass spectrometry, which relies on very high mass accuracy of the instruments, specific ionization patterns during mass fragmentation, as well as multiple stringent steps of automated and manual validation. Below we describe the methods of in vitro arginylation and mass spectrometry analysis of arginylated proteins, developed by our groups during the last 15 years.

1. Introduction

Posttranslational protein modifications by covalent addition of various chemical groups to proteins play key roles in normal physiology, and contribute to a large number of disease states. While hundreds of possible post-translational modifications have been identified, for most of them we still know very little about their molecular mechanisms, as well as their downstream effects protein properties and functions. Thus, studies of posttranslational modifications and their biological roles constitute a rapidly emerging important future direction that will provide critical insights into our understanding of life.

Protein arginylation—enzymatic addition of the amino acid arginine (Arg) to proteins, mediated by arginyltransferase ATE1—has been discovered in 1963 (Kaji, 1968; Kaji, Novelli, & Kaji, 1963), but is still relatively poorly understood. ATE1 utilizes charged Arg-tRNA as the donor of the Arg moiety (Kaji, 1968; Kaji, Kaji, & Novelli, 1963, 1965a, 1965b; Kaji & Rao, 1976), reminiscent of conventional translation, but independently of the ribosome. Initial work characterized arginylation as an exclusively N-terminal modification that targets N-terminally exposed acidic residues, Asp or Glu. Later on, N-terminally exposed Cys was added to this list, but Cys arginylation has only been shown to occur in mammalian cells (Gonda et al., 1989; Varshavsky, 1992; Varshavsky, 1995). More recently, it was discovered that Cys arginylation is greatly facilitated by its trioxidation, and this has been proposed to serve as an important mechanism of oxygen sensing in cells (Dissmeyer, 2019; Hu et al., 2005; Lee et al., 2005; Wang et al., 2018; White et al., 2017).

Methodological breakthroughs in biochemical analysis of arginylation, including establishment of a reliable in vitro arginylation system, and a high throughput method of arginylation identification by mass spectrometry (both described in this chapter), greatly expanded our understanding of arginylation. These new studies enabled identification of additional 100+ in vivo targets of arginylation (Karakozova et al., 2006; Rai et al., 2008; Wong et al., 2007; Xu et al., 2009) and enabled deeper insights into arginylation. It was found that ATE1 is capable of self-arginylation (Wang et al., 2011), and that in vivo arginylated proteins can further undergo mono- or dimethylation on the added Arg (Saha et al., 2011). Surprisingly, these studies also showed that, in addition to the previously described N-terminal chemistry, proteins can be arginylated on side chains of Asp/Glu, forming an isopeptide bond with the Arg amino group (Eriste et al., 2005; Wang et al., 2014). A number of “non-canonical” N-terminal arginylation sites, with Arg added different amino acid residues, have been identified in broad mass spectrometry searches (Wong et al., 2007), even though it is still not clear whether all of these cases are ATE1-mediated, or occur via another as yet unidentified arginylation activity in cells. All these discoveries have significantly broadened the scope of potential in vivo effects of ATE1.

ATE1 is present in multiple eukaryotic organisms, from yeast to humans, and has been demonstrated early on to occur in plants (Manahan & App, 1973), correlate with mammalian cell aging (Lamon & Kaji, 1980), and increase during nerve regeneration after injury (Wang & Ingoglia, 1997; Xu et al., 1993), and oxidative stress (Zhang, Donnelly, & Ingoglia, 1998). Mouse knockout of ATE1 leads embryonic lethality and severe defects in cardiovascular development and angiogenesis (Kwon et al., 2002). ATE1 is essential for viability of animal organisms starting with flies (reviewed in Saha & Kashina, 2011). In mice, ATE1 deletion in different tissues and organ systems demonstrates its essential role in neural crest-dependent craniofacial morphogenesis (Kurosaka et al., 2010), the formation and contractility of the cardiac muscle (Rai et al., 2008), and gametogenesis (Leu, Kurosaka, & Kashina, 2009). Postnatal deletion of ATE1 leads to weight loss, mental retardation, and infertility (Brower & Varshavsky, 2009). ATE1 deletion in Arabidopsis thaliana results in delayed leaf senescence (Lim, Kim, & Nam, 2007; Yoshida et al., 2002), defective shoot and leaf development (Graciet et al., 2009) and abnormal seed germination (Holman et al., 2009). To date, hundreds of proteins arginylated in vivo have been identified, and this list is growing by the day.

2. Methodological approaches to protein arginylation analysis

Studies of posttranslational modifications present many technical challenges, which are different for every modification studied. In the case of arginylation, the difficulties arise from the fact that Arg is a regular amino acid that also incorporates into proteins during translation. Thus, in vitro arginylation needs to be conducted in a strictly ribosome-free system, while in vivo identification of arginylated proteins by mass spectrometry has to rely on very high mass accuracy of the instrument, specific ionization patterns during mass fragmentation, as well as multiple stringent steps of automated and manual validation. These methods are labor intensive, but well developed, as described in the chapter below.

Additional methodological approaches to the studies of arginylation are possible in principle, and need to be considered by anyone venturing out into arginylation studies. One strategy involves development of antibodies specific to arginylated proteins. This strategy is feasible based on the success with similar approaches in the studies of other posttranslational modifications (e.g., phospho-specific antibodies). However, arginylation meets with the unique challenge because of the high similarity of the added Arg to those found in the protein backbone. Thus, each of these antibody projects needs a carefully planned design.

In the past, we have been partially successful with developing “panarginylation” antibodies, designed to recognize any protein with N-terminal Arg followed by either Asp of Glu, the preferred target sites for arginylation (Wong et al., 2007). A similar approach to the design of mid-chain-arginylated sites (with Arg attached to the carboxy side chain of Asp or Glu via isopeptide bond), and/or to the added mono- or dimethylated Arg, should be potentially even more effective.

Peptide antibodies to individual arginylated proteins is a more straight-forward approach, useful when addressing the biological role of arginylation on highly promising protein targets. EMD Millipore has developed the first successful antibody to N-terminally arginylated beta actin (catalog # ABT264), which was validated in our recently published studies (Pavlyk et al., 2018; Wang et al., 2017). EMD Millipore also carries a rat monoclonal anti-ATE1 developed by our lab (Wang et al., 2011), which is highly specific, and has broad reactivity with ATE1 in different species (EMD Millipore catalog # MABS436).

Promising future approaches to arginylation detection involve developing chemical analogs of Arg and tRNA, which cannot be utilized by the ribosome but can potentially be recognized by ATE1. Such an approach has been successfully employed with L/F transferase, the bacterial enzyme analogous to ATE1 that transfers Leu and Phe onto the N-terminal Arg (Leibowitz & Soffer, 1969). This enzyme can utilize aminoacyl adenosine in place of tRNA as the carrier of donor amino acids (Tanaka et al., 2013; Wagner et al., 2011), providing a highly specific way of separating the modifications by L/F transferase from regular translation. In principle, if a similar chemical tRNA mimic could be found that is compatible with arginyl transfer reaction, it could become a powerful tool in arginylation studies.

3. Bacterial expression and purification of recombinant mouse arginyltransferase (ATE1) and human Arg-tRNA synthetase (RRS) for arginylation assays

Here we describe the procedure for expression and purification of recombinant mouse ATE1 and human RRS. This method is easy and convenient, and can result in one-step isolation of milligram amounts of soluble enzymatically active ATE1 or RRS at nearly 99% purity (Wang & Kashina, 2015a, 2015b; Wang et al., 2011).

3.1. Equipment

  1. Amerex Gyromax 737 incubator shaker for bacterial growth.

  2. Precision Scientific 360 Orbital Shaker Bath.

  3. Fisher Scientific Model 550 Sonic Dismembrator for bacterial cell lysis.

  4. Dupont Sorvall RC-5B Refrigerated Superspeed Centrifuge.

3.2. Materials

3.2.1. E. coli strains and plasmids

  1. BL21-CodonPlus® (DE3)-RIL and BL21(DE3) (Stratagene, USA) (Tip 1).

  2. Plasmids of pET29a (Novagen) with inserted cDNA encoding individual mouse ATE1 isoforms (Tip 2). ATE1 from other species can be used; we have previously tested yeast ATE1, in addition to the mammalian ATE1 isoforms.

  3. Plasmid pM368 encoding human RRS (kindly provided by Dr. Ya-Ming Hou, Thomas Jefferson University). RRS from other species can be used; we have previously tested E. coli RRS, in addition to the human enzyme.

3.2.2. Buffers, solutions and kits

  1. LB media supplemented with 100mg/mL kanamycin and 50mg/mL chloramphenicol (for ATE1) or 100mg/mL ampicillin (for RRS).

  2. 1M IPTG (isopropyl-1-thio-d-galactopyranoside) stock.

  3. Lysis Buffer: 0.5M NaCl, 1mM MgCl2, 50mm Tris, 10mM β-mercaptoethanol, 5mM imidazole, 1mM PMSF, pH 7.5.

  4. Wash Buffer: 1M NaCl, 1mM MgCl, 50mm Tris, 10mM β-mercaptoethanol, 25mM imidazole, pH 7.5.

  5. Elution Buffer: 0.5M NaCl, 1mM MgCl2, 50mM Tris, 10mM β-mercaptoethanol, 0.5M imidazole, pH 7.5.

  6. Dialysis Buffer: 25mM HEPES, 1mM DTT, 0.5M NaCl, 0.1mM EDTA, 1mM MgCl2, 50% glycerol, pH 7.5.

  7. Ni-NTA Agarose (QIAGEN catalog # 30210).

  8. Pierce™ BCA Protein Assay Kit (catalog # 23227).

3.3. Protocol

  1. Transform the E. coli BL21-CodonPlus® (DE3)-RIL competent cells with the ATE1 or RRS constructs following the manufacturer’s protocol.

  2. Grow an overnight 5mL culture of the transformed E. coli cells using Amerex Gyromax 737 Incubator Shaker, and inoculate the culture into 1L LB supplemented with 100mg/mL kanamycin and 50mg/mL chloramphenicol (for ATE1) or 100mg/mL ampicillin (for RRS).

  3. Continually grow the culture with shaking at 37°C until OD600 reaches 0.4–0.5.

  4. Cold-shock the culture by placing the entire flask on ice for 30min.

  5. For ATE1 expression: induce the culture by addition of 0.4mM IPTG and incubate for approximately 18h at 16°C on a Precision Scientific 360 Orbital Shaker Bath. For RRS expression: induce the culture by addition of 0.2mM IPTG and incubate for approximately 18h at 37°C on a shaker.

  6. Collect the cells by centrifuging at 5000g for 30min. Discard the supernatant.

  7. Resuspend the cell pellet in 10mL Lysis Buffer and sonicate the cells on ice using Fisher Scientific Model 550 Sonic Dismembrator at level 5, 6 × 10s with 1min intervals.

  8. Centrifuge the cell lysate at 30,597 g for 30min at 4°C. Discard the pellet.

  9. Load the supernatant onto the Ni-NTA agarose column pre-equilibrated with 10 column volumes of Lysis Buffer.

  10. Wash the column with 10 column volumes of Wash Buffer.

  11. Elute the protein with Elution Buffer, collecting fractions.

  12. Check the purity of the eluted fractions by 10% SDS-PAGE. The fractions should contain one major band running at ~60kDa, corresponding to the ATE1 protein or ~75kDa corresponding to human RRS (note: E. coli RRS runs at ~60kDa, similar to ATE1).

  13. Combine the peak fractions and dialyze against Dialysis Buffer overnight at 4°C.

  14. Determine the final protein concentration using Pierce™ BCA Protein Assay Kit following the manufacturer’s protocol.

  15. Aliquot the dialyzed protein into 1.5mL Eppendorf tubes and keep them at −80°C. The working fraction can be kept at −20°C for at least 2 weeks (see Tip 3).

3.3.1. Tips

  1. CodonPlus cells are necessary for the expression of mammalian ATE1 isoforms in E. coli.

  2. Tagging the ATE1’s N-terminus should be avoided, since the N-terminal portion of ATE1 has been found to be essential for its enzymatic activity (Rai & Kashina, 2005; Rai et al., 2006). N-terminal tagging works fine for RRS.

  3. 50% Glycerol introduced into this fraction during dialysis would prevent it from freezing solid at −20°C.

4. Assaying ATE1 activity in vitro

Here we describe a standard arginyltransferase assay in vitro using bacterially expressed purified ATE1 in a system with minimal number of components (Arg, tRNA, RRS, and arginylation substrate). Assays of this type have first been developed in 1980s using crude ATE1 preparations from cells and tissues (Ciechanover et al., 1988), and then optimized and improved recently by our group for the use with bacterially expressed recombinant proteins. This assay represents a simple and efficient way to measure ATE1 activity or to test its ability to arginylate particular protein and peptide substrates (Wang et al., 2011, 2014).

4.1. Equipment

  1. Denville Scientific IncuBlock

  2. Thermo Scientific Microcentrifuge

  3. Beckman Coulter LS6500 Multipurpose Scintillation Counter

4.2. Materials

  1. ATE1 and RRS obtained as described in the section above.

  2. 4× Assay Buffer: 200mM HEPES, 100mM KCl, 60mM MgCl2,0.4mM DTT, pH 7.5.

  3. Arginylation substrates. In our assays, we have successfully used bovine serum albumin (BSA), α-lactalbumin or α-synuclein as protein Arg acceptors (Section 4.3.1), and angiotensin II (DRVYIHPF, Section 4.3.2) as a peptide acceptor. Other synthetic peptides with N-terminal Asp or Glu can be used as peptide Arg acceptors and tend to incorporate Arg efficiently in ATE1-dependent reaction in vitro. Any other proteins or peptides to be tested as potential Arg acceptors can in principle be used as arginylation substrates in this assay (Wang et al., 2011, 2014).

  4. 100mM ATP stock.

  5. Peptide Wash Buffer: 0.1% trifluoroacetic acid (TFA) in water (Section 4.3.2).

  6. Peptide Elution Buffer: 60% acetonitrile, 0.1%TFA in water (Section 4.3.2).

  7. Labeled Arg: l-[2,3,4–3H]-Arg (PerkinElmer) for detection by scintillation counting; l-[14C(U)]-Arg (Moravek Biochemicals) for detection by autoradiography; or unlabeled l-Arg (Sigma) or l-[13C,15N]-Arginine (Pierce) for detection by mass spectrometry. Radioactively labeled Arg typically comes in solution. Non-radioactive Arg variants usually come dry and should be dissolved in water to 100mM prior to the experiment.

  8. tRNAArg (Arg-specific tRNA). Our data suggest that any tRNAArg from any species should be compatible with either mammalian or E. coli ATE1. If unavailable, bulk E. coli tRNA from Sigma, Roche, or other vendors can be used; however, this bulk tRNA has significantly lower efficiency in arginylation assays due to lower abundance of tRNAArg species.

  9. Trichloroacetic acid (TCA; for protein substrate precipitation, Section 4.3.1).

  10. BioPureSPN Mini, PROTO 300 C18 spin columns (The Nest Group, Inc) (for peptide substrate purification, Section 4.3.2).

  11. Acetonitrile (for peptide substrate purification, Section 4.3.2).

  12. Ecoscint ORIGINAL scintillation solution (National Diagnostics).

4.3. Protocol

4.3.1. Arginylation assay using a protein ATE1 substrate

  1. On ice, mix a 50μL reaction containing 1 × Assay Buffer, 2.5mM ATP,12.5μM l-Arg (labeled or unlabeled for different detection methods, see Section 4.2., p. 7 above), 12.5μM tRNAArg, 1.35μM RRS,1.35μM ATE1, and 13.5μM protein substrate. Keep the reaction on ice until ready.

  2. On ice, mix the control reactions: two with all the above components excluding ATE1, one with all the above components excluding RRS, one with all the above components excluding protein substrate.

  3. Prepare quenching tubes, one for each time point (see below): 40μL of 20% trichloroacetic acid (TCA) containing 1mM of unlabeled Arg.

  4. When all the reactions are mixed, take a 10μL aliquot from each tube (0 time point) and immediately quench it into the quenching tube to stop the reaction. Place all the tubes with the remaining mixtures simultaneously into a heat block pre-equilibrated to 37°C, and start the timer.

  5. Take out a 10μL aliquot at each time point (usually 10, 20, 30, and 40min) and immediately quench it into the quenching tube. The quenched aliquots can be kept at room temperature for 10min or so.

  6. Set aside one of the control sets containing no ATE1 and intact leftover Arg-tRNA. Heat the rest of the quenched aliquots at 95°C for 15min to destroy the leftover labeled Arg-tRNA.

  7. Put all the tubes on ice and keep them for 20min to cool down, then spin at 16,000g for 30min at room temperature to collect the pellets containing precipitated proteins.

  8. Wash the pellets three times by adding 5% cold TCA without disturbing the pellet and re-centrifuging at 16,000g for 10min at room temperature. Repeat the wash one more time using cold acetone in place of TCA. Air-dry the pellets.

  9. For detection by scintillation counting (l-[2,3,4–3H]-Arg), put the air-dried tubes (with open or removed caps) into scintillation vials filled with Ecoscint ORIGINAL scintillation solution and count with the Beckman Coulter LS6500 Multipurpose Scintillation Counter. ATE1 activity (or charged Arg-tRNA levels for unheated control) will be measured as counts per minute (cpm) of [3H] at each time point.

For arginylation detection by autoradiography (l-[14C(U)]-Arg), instead of TCA quenching, samples at the end point should be mixed with an equal volume of 2× SDS sample buffer and boiled, then separated on the SDS page, dried, and exposed to X-ray film at −80°C (usually for several days or longer). Since 14C is very stable, gels could be stored and re-exposed indefinitely.

For detection of in vitro arginylated proteins by mass spectrometry (described below), unlabeled Arg or [13C,15N]-labeled Arg should be used in the reaction. The use of heavy isotope-labeled Arg in parallel with unlabeled one can provide an independent control and thus increase the confidence of the assay. Instead of time points, samples should be arginylated for 40min, precipitated with 20% TCA and processed as described elsewhere (Wong et al., 2007; Xu et al., 2009) and in the protocol below.

4.3.2. Arginylation assay using a peptide ATE1 substrate

  1. On ice, mix a 100μL reaction containing 1 × Assay Buffer, 2.5mM ATP, 12.5μM tRNAArg, 1.35μM RRS,1.35μ M ATE1, 15μM peptide substrate, and 12.5μM Arg (3H-labeled for scintillation counting, unlabeled or [13C,15N]-labeled Arg for mass spectrometry detection).Simultaneously, set up the control reactions as described in Section 4.3.1.

  2. Place the reactions into a pre-equilibrated 37°C heat block and incubate for 15min.

  3. Terminate the reactions by heating at 95°C for 15min.

  4. Keep the tubes on ice for 20min, and then spin at 16,000g for 15min at room temperature.

  5. Load the supernatants onto BioPureSPN Mini, PROTO 300 C18 spin columns prewashed with 100% acetonitrile and water by centrifugation at 110g for 1 min.

  6. Wash the columns with 150μL Peptide Wash Buffer by centrifugation at 110g for 1 min.

  7. Elute the columns with 150μL Peptide Elution Buffer by centrifugation at 110g for 1min. For radioactive Arg, eluted peptides at this stage should be transferred into scintillation vials and analyzed on a liquid scintillation counter. For mass spectrometry, steps 8 and 9 below should be performed.

  8. Dry the eluted peptides using speed vacuum until the disappearance of any visible liquid from the tube. The peptide should form a thin film-like pellet that may not be clearly visible.

  9. Re-dissolve the arginylated peptides in Peptide Wash Buffer for further analysis by mass spectrometry.

5. Identification of arginylated proteins by mass spectrometry

This method has been under development since 2006, and has evolved with the changes in our understanding of arginylation. The original version of this method has been applied to identification of N-terminal arginylation without consideration of the target residue (Wong et al., 2007; Xu et al., 2009). With the discovery of side chain arginylation (Wang et al., 2014) we have used a different search algorithm to search only for the addition of Arg to Asp or Glu, independent of their position in the peptide. In principle, this search resembles searches for other posttranslational modifications (e.g., phosphorylation), except that the mass of Arg is used. Given the relatively high molecular weight of Arg, this analysis requires the use of the mass spectrometry instruments capable of very high mass accuracy (LTQ-Orbitrap), as well as extra stringent automated data filtering, followed by manual elimination of mass ambiguities (Tables 1–3) and manual validation of the identified spectra. This method can be used with both complex and purified protein samples and, to date, constitutes the only reliable way to confirm arginylation at a particular site on a protein or peptide.

Table 1.

Mass ambiguities for arginylated peptides.

Unmodified ARG (156.1011)
−2 residue −1 residue −2 residue mass −1 residue mass Posttranslational Modification (PTM) PTM mass shift Residue(s) + PTM mass Delta Mass (amu) Delta Mass in ppm ppm delta mass for peptides of mass 1000 amu ppm delta mass for peptides of mass 2000 amu ppm delta mass for peptides of mass 3000 amu
V G 99.0684 57.0215 None 156.0899 −0.0112 −71.9534 −11.2320 −5.6160 −3.7440
A G 71.0371 57.0215 Formylation 27.9949 156.0535 −0.0476 −305.1356 −47.6320 −23.8160 −15.8773
A G 71.0371 57.0215 Di-methylation or ethylation 28.0313 156.0899 −0.0112 −71.9534 −11.2320 −5.6160 −3.7440
G A 57.0215 71.0371 Formylation 27.9949 156.0535 −0.0476 −305.1356 −47.6320 −23.8160 −15.8773
G A 57.0215 71.0371 Di-methylation or ethylation 28.0313 156.0899 −0.0112 −71.9534 −11.2320 −5.6160 −3.7440
G V 57.0215 99.0684 156.0899 −0.0112 −71.9534 −11.2320 −5.6160 −3.7440
V 99.0684 Non-specific alkylation 57.0215 156.0899 −0.0112 −71.9534 −11.2320 −5.6160 −3.7440
L/I 113.0841 Carbamylation 43.0058 156.0899 −0.0113 −72.0687 −11.2500 −5.6250 −3.7500
N 114.0429 Acetylation 42.0106 156.0535 −0.0476 −305.0267 −47.6150 −23.8075 −15.8717
N 114.0429 Guanidination 42.0218 156.0647 −0.0364 −233.0669 −36.3820 −18.1910 −12.1273
N 114.0429 Tri-methylation 42.0470 156.0899 −0.0112 −71.9406 −11.2300 −5.6150 −3.7433
D 115.0269 Amidine 41.0265 156.0535 −0.0476 −305.0651 −47.6210 −23.8105 −15.8737
Q 128.0586 Di-methylation or ethylation 28.0313 156.0899 −0.0112 −71.9406 −11.2300 −5.6150 −3.7433
R 156.1011 None 156.1011 0.0000 0.0000 0.0000 0.0000 0.0000
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Mass shifts equal to Arg addition (+156.1011Da) produced by different amino acid residues and posttranslational modifications in the positions adjacent and preceding the arginylated site. These ambiguities cannot be resolved by mass spectrometry and must be manually discarded.

Delta mass=residue(s)+PTM mass shift−156.1011.

Delta mass in ppm=delta mass/156.1011*1,000,000.

ppm delta mass for peptides with mass of 1000=delta mass/1000*1,000,000.

ppm delta mass for peptides with mass of 1000=delta mass/2000*1,000,000.

ppm delta mass for peptides with mass of 1000=delta mass/3000*1,000,000.

Table 3.

Mass ambiguities for dimethyl-arginylated peptides.

Dimethylated ARG (184.1325)
−2 residue −1 residue −2 residue mass −1 residue mass Posttranslational Modification (PTM) PTM mass shift Residue(s) + PTM mass Delta Mass(amu) Delta Mass in ppm ppm delta mass for peptides of mass 1000 amu ppm delta mass for peptides of mass 2000 amu ppm delta mass for peptides of mass 3000 amu
*G A 57.02146 71.03711 Diethylation 56.06266 184.1212 −0.01117 −60.6737 −11.172 −5.586 −3.724
*G P 57.02146 97.05276 Hydroxymethyl 30.01057 184.0848 −0.04762 −258.602 −47.617 −23.8085 −15.8723
*G V 57.02146 99.06841 Di-methylation or ethylation 28.0313 184.1212 −0.01123 −60.9996 −11.232 −5.616 −3.744
*G I/L 57.02146 113.0841 Methylation 14.0157 184.1212 −0.01119 −60.7498 −11.186 −5.593 −3.72867
*G V 57.02146 99.06841 Dimethylation/ethylation 28.0313 184.1212 −0.01123 −60.9996 −11.232 −5.616 −3.744
*G Q 57.02146 128.0586 Amidation −0.9848 184.0952 −0.03717 −201.844 −37.166 −18.583 −12.3887
*G K 57.02146 128.095 Amidation −0.9848 184.1317 −0.00075 −4.05143 −0.746 −0.373 −0.24867
A A 71.03711 71.03711 Acetylation 42.01057 184.0848 −0.04762 −258.602 −47.617 −23.8085 −15.8723
A P 71.03711 97.05276 Oxidation/hydroxylation 15.9949 184.0848 −0.04763 −258.683 −47.632 −23.816 v15.8773
A V 71.03711 99.06841 Methylation 14.0157 184.1212 −0.01118 −60.728 −11.182 −5.591 −3.72733
A N 71.03711 114.0429 Amidation −0.9848 184.0952 −0.0372 −202.007 −37.196 −18.598 −12.3987
A E 71.03711 129.0426 Deoxy −15.9949 184.0848 −0.04761 −258.542 −47.606 −23.803 −15.8687
A M 71.03711 131.0405 Oxoanaline −17.9928 184.0848 −0.04762 −258.597 −47.616 −23.808 −15.872
S P 87.03203 97.05276 184.0848 −0.04762 −258.602 −47.617 −23.8085 −15.8723
S I/L 87.03203 113.0841 Deoxy −15.9949 184.1212 −0.01122 −60.9398 −11.221 −5.6105 −3.74033
P C 97.05276 103.0092 Deoxy −15.9949 184.0671 −0.06536 −354.94 −65.356 −32.678 −21.7853
V T 99.06841 101.0477 Deoxy −15.9949 184.1212 −0.01122 −60.9127 −11.216 −5.608 −3.73867
Q 128.0586 Diethylation 56.06266 184.1212 −0.01117 −60.6629 −11.17 −5.585 −3.72333
K 128.095 Diethylation 56.06266 184.1577 0.02525 137.1296 25.25 12.625 8.416667
W 186.0793 Ddehydro −2.01565 184.0637 −0.06875 −373.373 −68.75 −34.375 −22.9167
R 156.1011 Dimethylation/ethylation 28.0313 184.1324 0 0 0 0 0
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*

G (57.0215) can be either G or alkylation.

Mass shifts equal to monomethylated Arg addition (+184.1325Da) produced by different amino acid residues and posttranslational modifications in the positions adjacent and preceding the arginylated site. These ambiguities cannot be resolved by mass spectrometry and must be manually discarded.

Mass difference=residue(s)+PTM mass shift−184.1325.

Mass difference in ppm=Mass difference/184.1325 *1,000,000.

5.1. Equipment

  1. Mass spectrometer (LTQ-Orbitrap, Thermo Fisher Scientific, or equivalent, capable of similar or higher mass accuracy.)

  2. Software (SEQUEST, ProLuCID, DTASelect 2.0, deltaMassFilter)

5.2. Materials (see Tip 1)

  1. Protein or peptide sample for analysis (see Tip 2).

  2. Protein digestion solution: 1mg/mL trypsin (mass spectrometry grade, Promega, Cat#V5280) in 50mM ammonium bicarbonate and 5mM CaCl2 (store in aliquots at −20°C; each aliquot after thawing can be used once) (see Tip 3 for other protease options).

  3. Trichloroacetic Acid (TCA) (Sigma, Cat#T9159) (see Tip 4).

  4. Acetone (Thermo Fisher, Cat#BP2403-4) (stored at −20°C).

  5. Invitrosol LC/MS protein solubilizer (Invitrogen, Cat #MS10007).

  6. Protein alkylation solution: 500mM iodoacetamide (Sigma Aldrich, Cat #I1149) in 100mM ammonium bicarbonate (Sigma Aldrich, Cat #11213) and 10mM Tris (2-carboxyethyl)phosphine hydrochlo-ride (Sigma Aldrich, Cat #C4706), pH 8.5 (freshly made).

  7. Peptide extraction buffer: 5% formic acid (J.T. Baker, Cat#0129-01) (can be stored at room temperature for 1–4 months).

  8. Buffer A (peptide loading buffer for liquid chromatography MS/MS): 5% acetonitrile (Sigma Aldrich, Cat#00683), 0.1% formic acid in water (can be stored at room temperature for 1–4 months).

  9. Buffer B (peptide elution buffer for liquid chromatography MS/MS): 100% acetonitrile, 0.1% formic acid in water (can be stored at room temperature for 1–4 months).

  10. Buffer C (alternative peptide elution buffer for liquid chromatography MS/MS, see Note 10): 500mM ammonium acetate, 100% acetonitrile, 0.1% formic acid in water (can be stored at room temperature for 1–4 months).

  11. Calcium chloride (Sigma Aldrich, Cat #12022).

  12. Ammonium acetate (Sigma Aldrich, Cat#73594).

  13. 1M Dithiothreitol (DTT, Sigma Aldrich, Cat# 43815) in water (stored at −20°C).

5.3. Protocol

5.3.1. Sample preparation and mass spectrometry

  1. Add 1:10 volume of 100% TCA solution to the sample.

  2. Vortex briefly and incubate on ice for 30min.

  3. Centrifuge at 13000g for 10min. Remove the supernatant.

  4. Acetone wash (repeat three times): add 100% cold acetone to the protein pellet, centrifuge at 13000g for 10min, remove the acetone.

  5. Remove the acetone and air-dry the pellet (see Tip 5).

  6. Dissolve the pellet in the protein solubilizer.

  7. Disulfide bond reduction: add DTT to the final concentration of 10mM and incubate 30min at room temperature.

  8. Alkylation: Add protein alkylation solution to the final concentration of 55mM and incubate 20min at room temperature in the dark (see Tip 6).

  9. Add protease solution (trypsin or another protease, see Tip 3) to the protein sample at 1:100 weight ratio (i.e., 1μg of trypsin per 100μg of protein in the sample) (see Tip 7). Digest for 1h at 37°C. Longer digestion times, up to overnight, and higher enzyme ratios (e.g., 1:20 for LysC and 1:50 for trypsin) can be used with higher complexity samples or less efficient enzymes.

  10. Stop the digestion by adding of 5% formic acid to the protein digestion solution. Collect the soluble fraction containing the extracted peptides (see Tips 7 and 8).

  11. Load the sample onto a reverse phase (RP) or strong cation exchange/RP MudPIT column and elute with a linear 5–100% gradient of acetonitrile (see Tip 9).

5.3.2. Identification of arginylated peptides in the samples

  1. Analyze the results by database searching. For N-terminal Arg identification use ProLuCID (Xu et al., 2006) or an equivalent program (see Tip 10). For side chain Arg identification, use the differential modification search with or without addition of 156.1011 (Arg), 170.1168 (monomethylated Arg), 184.1325 (dimethylated Arg) on every Asp and Glu. The search results will yield the initial list of putative arginylated peptides (see Tip 11).

  2. Use DTASelect 2.0 algorithm (Cociorva, Tabb, & Yates, 2007; Shen et al., 2008; Tabb, McDonald, & Yates, 2002) for automated data filtering of the search to eliminate the initial set of the false positives (see Tip 12).

  3. Use deltaMassFilter on the data from step 2 for high mass accuracy filtering. Use the p-value setting −p 0.0001 (see Tip 13).

  4. Manually analyze the identified arginylated peptides for possible mass ambiguities listed in Tables 1–3. Discard the peptides, for which mass ambiguities cannot be resolved even with the high mass accuracy data (e.g., preceding R, preceding I/L if urea was used in the sample, preceding V, and preceding GV/VG sequence).

  5. Validate the arginylated peptide spectra manually by isotopic peak checking (see Tip 14).

  6. Check the ion fragmentation (MS/MS) spectrum for arginylated peptides in the Xcalibur Raw file using the Qual Browser program by manual viewing to identify the b ion series. Addition of N-terminal Arg onto peptides is expected to result in an altered pattern of fragment ions generated from the peptide’s N-terminus (b ion series). Arginylated peptides usually have more prominent early b ions (corresponding to the lower mass fragments generated from the N-terminus) (Dongre et al., 1996; Karakozova et al., 2006; Tsaprailis et al., 1999; Wong et al., 2007).

5.3.3. Tips

  1. The specific reagent grade, as listed, is important for this procedure.

  2. It is recommended to analyze proteins in solution, rather than in-gel, to achieve higher sequence coverage and minimal interference from possible chemical modifications during sample preparation. The probability of detection of arginylated peptides increases with higher protein abundance and lower protein complexity. Some purification methods may either enrich or select against the arginylated protein in the preparations. The use of urea (e.g., during protein solubilization or 2D gel fractionation) or Gly (common in buffers, e.g., SDS PAGE running buffer) should be avoided during sample preparation, as they can result in the following mass ambiguities: carbamylation of Leu (by urea, making Leu similar in mass to Arg) and non-specific glycylation of the free amino groups of the protein’s N-terminus (by Gly in the buffer, which may alter the apparent mass of the adjacent Val to mimic the mass of Arg).

  3. Choice of proteases is critically important for this analysis. While use of multiple proteases (for example, a combination of trypsin, subtilisin, and elastase) can increase sequence coverage during mass spectrometry, some of these proteases may destroy arginylated peptides by cutting immediately after the arginylated residue or producing peptides too short for detection. We found that trypsin is the optimal enzyme (highly efficient and enabling the maximum sequence coverage during identification for the majority of proteins). While its specificity, in cutting C-terminally to Arg and Lys, may potentially remove the added Arg through exopeptidase activity, we found no such activity in our control tests using standard synthetic peptides, and have routinely identified multiple arginylated proteins in trypsin-digested samples. Thus, we typically use trypsin digestion for the analysis of arginylation.

  4. TCA should not be stored dry, as it absorbs water and thus skews the preparation of TCA solutions after storage. Prepare 100% solution immediately after arrival by adding water to the TCA jar so that the final volume of the solution equals the weight of TCA in the jar, e.g. 250mL for 250g package of TCA. Store at 4°C.

  5. TCA-precipitated protein pellet can be stored at −20°C for up to several months.

  6. Iodoacetamide is highly unstable and light sensitive. Solution needs to be made fresh and protein alkylation needs to be performed in the dark.

  7. Longer digestion times, up to overnight, and higher enzyme ratios can be used with higher complexity samples and lower efficiency proteases. Microgram quantities of the samples are recommended for best detection.

  8. Digested peptide solution at this stage can be concentrated by evaporation on the Speedvac until only the insoluble peptide pellet remains and redissolving the pellet in 5% acetonitrile:0.1% formic acid. This step can improve the analysis but is not essential. Digested peptides at this stage can also be frozen and stored, if needed.

  9. For single proteins and low complexity samples a one-step acetonitrile gradient is typically used. For the analysis of complex samples using Multidimensional Protein Identification Technology (MudPIT (Washburn, Wolters, & Yates, 2001)) a 12-step gradient is recommended, as follows:

    Steps 1–11:
    • 1min of 100% buffer A
    • 5min of X% buffer C (the 5min buffer C percentages (X) are 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%)
    • a 13min 0–15% gradient of buffer B
    • a 107min 15–45% gradient of buffer B
    • a 30min 45–75% gradient of buffer B
    • a 5min 75–100% gradient of buffer B
    • 5–20min of 100% buffer A.
    Step 12:
    • 3min of 100% buffer A
    • 20min of 100% buffer C
    • a 10min 0–15% gradient of buffer B
    • a 107min 15–70% gradient of buffer B

  10. In principle any commercially available and/or user-developed programs for identifying of the addition of a fixed mass on a peptide’s N-terminus and/or Asp/Glu could be used, such as SEQUEST (Eng, McCormack, & Yates, 1994), Mascot (Perkins et al., 1999), X!Tandem (Craig & Beavis, 2004), or OMSSA (Geer et al., 2004), but many factors should be accounted for in the development and use of such programs, and the validation steps described below should always be performed on the list of initial positives to identify the true arginylation targets. Based on our experience, ProLuCID (Xu et al., 2006) that we use in our analysis has better sensitivity and specificity for both modified and non-modified peptide identification compared to SEQUEST, Mascot, X!Tandem, and OMSSA. Thus, we recommend ProLuCID over other algorithms.

  11. Depending on the sample preparation conditions, static modifications should be added into the search, e.g., for Cys in case of treatment with a reducing reagent (+57.02146).

  12. For DTASelect2.0 filtering we use false positive rate parameter (fp) defined as the ratio of the number of reverse (i.e., false) hits to the number of forward (i.e., true) hits that passed the DTASelect filtering. We filter with the false positive rate setting of 0.1% (fp 0.001). To ensure that the false positive rate is constant in the searches for modified and unmodified peptides, DTASelect2.0 applies separate filtering to modified and unmodified peptides, considering the possibilities of tryptic, half tryptic and non-tryptic peptides (–modstat and –trypstat options of DTASelect2.0). One peptide serves as a requirement for the identification of a protein (−p 1) since we are looking for a specific modification feature of the protein rather than protein identification in the sample. Note that the high accuracy precursor mass information is not used in this step.

  13. The deltaMassFilter program can effectively remove most simple ambiguities that result in a significant mass shift. For this filtering, the p-value for each peptide precursor mass is calculated as the delta mass (defined as the difference between the measured mass and the theoretical mass) for each modified peptide against the distribution of the delta masses of all the non-modified peptides. For each modified peptide, the delta mass p-value defines the chance that the observed mass corresponds to this particular peptide and not a highly similar one created by a similar mass shift as a result of another modification. A true hit (i.e., truly arginylated peptide) is assumed to have delta mass distribution similar to the distribution of the unmodified peptides, as evidenced by sufficiently high p-value.

  14. For example, if the calculated mass (M) of the identified peptide is 1500, and the measured mass of the peptide is 1501, check the peak in the corresponding MS spectrum in the Raw file to confirm that the measured peak is indeed the M+1 peak, (which occurs if one of the carbons in the peptide is a heavy 13C). If the measured mass of the peptide is 1500, then confirm that the peak in the corresponding MS spectrum is a monoisotopic peak. If this cannot be confirmed, eliminate the corresponding peptides as false positives. Ultimately, a sequence assignment can be validated by synthesis of the peptide and comparison of the tandem mass spectra.

Table 2.

Mass ambiguities for monomethyl-arginylated peptides.

Monomethylated ARG (170.1168)
−2 residue −1 residue −2 residue mass −1 residue mass Posttranslational modification (PTM) PTM mass shift Residue(s) + PTM mass Delta Mass (amu) Delta Mass in ppm ppm delta mass for peptides of mass 1000 amu ppm delta mass for peptides of mass 2000 amu ppm delta mass for peptides of mass 3000 amu
A aG 71.0371 57.0215 Acetylation 42.0106 170.0691 −0.0477 −280.2016 −47.6670 −23.8335 −15.8890
A A 71.0371 71.0371 Di-methylation or ethylation 28.0313 170.1055 −0.0113 −66.3191 −11.2820 −5.6410 −3.7607
A V 71.0371 99.0684 170.1055 −0.0113 −66.3191 −11.2820 −5.6410 −3.7607
aG V 57.0215 99.0684 Methylation 14.0157 170.1056 −0.0112 −66.0252 −11.2320 −5.6160 −3.7440
aG L/I 57.0215 113.0841 170.1055 −0.0113 −66.3427 −11.2860 −5.6430 −3.7620
S T 87.03203 101.0477 Dehydration −18.0106 170.0691 −0.0477 −280.4014 −47.7010 −23.8505 −15.9003
S V 87.03203 99.0684 Deoxy −15.9949 170.1055 −0.0113 −66.2310 −11.2670 −5.6335 −3.7557
Q 128.0586 Acetylation 42.0106 170.0691 −0.0477 −280.1898 −47.6650 −23.8325 −15.8883
Q 128.0586 Guanidination 42.0218 170.0804 −0.0364 −214.1470 −36.4300 −18.2150 −12.1433
Q 128.0586 Tri-methylation 42.0470 170.1056 −0.0112 −66.0135 −11.2300 −5.6150 −3.7433
K 128.095 Acetylation 42.0106 170.1056 −0.0112 −66.1016 −11.2450 −5.6225 −3.7483
E 129.0426 Amidine 41.0265 170.0691 −0.0477 −280.2251 −47.6710 −23.8355 −15.8903
W 186.0793 Deoxy −15.9949 170.0844 −0.0324 −190.5455 −32.4150 −16.2075 −10.8050
R 156.1011 Methylation 14.0157 170.1168 0.0000 0.0000 0.0000 0.0000 0.0000
Open in a new tab
a

G (57.0215) can be either G or alkylation.

Mass shifts equal to monomethylated Arg addition (+170.1168Da) produced by different amino acid residues and posttranslational modifications in the positions adjacent and preceding the arginylated site. These ambiguities cannot be resolved by mass spectrometry and must be manually discarded.

Mass difference=residue(s)+PTM mass shift−170.1168.

Mass difference in ppm=Mass difference/170.1168*1,000,000.

ppm Mass difference for peptides with mass of 1000=Mass difference/1000*1,000,000.

ppm Mass difference for peptides with mass of 1000=Mass difference/2000*1,000,000.

ppm Mass difference for peptides with mass of 1000=Mass difference/3000*1,000,000.

The −1 and −2 residues can be swapped.

Acknowledgments

This work was supported by NIH grants R35 GM122505 and R01NS102435 to A.K. and P41 GM103533 to J.R.Y.

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