Synthesis of peptides and proteins with site-specific glutamate arginylation

Abstract

Solid-phase peptide synthesis and protein semi-synthesis are powerful methods for site-specific modification of peptides and proteins. We describe protocols using these techniques for the syntheses of peptides and proteins bearing glutamate arginylation (E^Arg) at specific sites. These methods overcome challenges posed by enzymatic arginylation methods and allow for a comprehensive study of the effects of E^Arg on protein folding and interactions. Potential applications include biophysical analyses, cell-based microscopic studies, and profiling of E^Arg levels and interactomes in human tissue samples.

1. Introduction

Protein arginylation is an understudied post-translational modification (PTM), catalyzed by arginyl-tRNA transferase enzyme 1 (ATE1), which transfers Arg from an aminoacyl tRNA to the protein N-terminus or to the side chain carboxylates of Asp or Glu[1]. To study the effects of this PTM, the Kashina group has developed enzymatic arginylation methods using purified ATE1 and arginylated tRNA or tRNA fragments[2–6]. However, the in vitro enzymatic modification protocols pose major challenges in terms of yield and purification. ATE1 does not appear to have tight control over site-specificity of the modification, and the level of the modification can be low especially for protein substrates[3] – typically there is a mixture of differentially arginylated substrates. This makes it very hard to isolate a desired modification product, as these substrates often coelute on column chromatography. Preparing proteins with homogenous N-terminal arginylation can be achieved relatively simply by recombinant expression of the target protein with a cleavable N-terminal tag to eventually expose an inserted, N-terminal arginine. Methods for studying N-terminal arginylation are discussed in, e.g., Chapters 8, 9, 10, 16, and 20 of this book. To generate proteins with side-chain arginylation homogenously incorporated, we developed a site-specific incorporation approach and performed semi-syntheses of alpha-synuclein (αS) with glutamate arginylation (E^Arg) at site 46, 83, or both, combined with an orthogonally installed fluorophore, which have been used to study the effects of the modification both in vitro[7] and in living cells[8, 9]. Additionally, we synthesized E^Arg peptides for generation of anti-arginylation antibodies, including those for specific E^Arg sites in αS [8] and pan-E^Arg antibodies (in progress) to assess global changes in arginylation.

Here, we describe protocols for synthesizing peptides and proteins bearing E^Arg at specific sites (Figure 1). These include the syntheses of the E^Arg monomer unit and peptides, followed by protein semi-synthesis. The initial step is the synthesis of the E^Arg monomer unit, which contains protecting groups needed for Fmoc-based solid-phase peptide synthesis (SPPS). We then describe the syntheses of four different types of peptides: antigen peptides for generating antibodies specific to a protein arginylation site (5); antigen peptide libraries for generating a pan- E^Arg antibody (7), which recognizes arginylation regardless of the surrounding sequence; photo-crosslinkable peptides for E^Arg-dependent interactome studies (9); and peptide fragments to incorporate the PTM for use in protein semi-synthesis (21).

Figure 1.

Applications of synthetic E^Arg peptides and proteins.

Semi-synthesis also involves cloning and the recombinant expression and purification of protein fragments for assembly through native chemical ligation (NCL)[10]. We included expression of a protein fragment bearing a “click” chemistry handle through unnatural amino acid mutagenesis[11] for site-specific fluorescence labeling, which can be combined with NCL for efficient multiple labeling of proteins, as we have shown repeatedly for αS[7, 12–15]. As the last step of semi-synthesis, NCL assembles peptide/protein fragments into a full-length protein, followed by in situ desulfurization, converting a thiol-containing amino acid to a native amino acid (i.e. cysteine to alanine, penicillamine to valine). Such a protein could be used in fluorescence correlation spectroscopy (FCS) to monitor the interaction of the protein with another protein or macromolecule[7, 15]. More complex adaptations of these methods can be used to introduce a second fluorophore for Förster resonance energy transfer (FRET) experiments to probe conformational change, which our laboratory has done with other PTMs, like tyrosine phosphorylation[15]. The types of experiments enabled by this fluorescent labeling have been reviewed elsewhere, in the context of amyloid forming proteins like αS[16], as well as for protein folding more generally[17].

Taken together, these methods allow for a comprehensive study of the effects of this PTM on protein folding and interactions, from detailed biophysical studies to cell-based microscopy studies and even profiling of E^Arg levels and interactions in human tissue samples. While the examples are all provided in the context of αS, the methods are general, and can be applied to any protein of interest that is amenable to semi-synthesis.

2. Materials

Use MilliQ filtered (18 MΩ) water (MilliporeSigma; Burlington, MA, USA) to make all the buffers.

2.1. Arginylated glutamate monomer synthesis

1. N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-glutamic acid 1-Allyl ester (Fmoc-Glu-OAll) (VWR; Radnor, PA, USA)

2. tert-Butyl N-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-L-argininatehydrochloride (H-Arg(Pbf)-OtBu•HCl) (VWR)

3. Isobutylchloroformate (IBCF) (Sigma-Aldrich; St. Louis, MO, USA)

4. N-Methylmorpholine (NMM) (Alfa Aeser; Tewksbury, MA, USA)

5. Phenylsilane (Sigma-Aldrich)

6. Tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) (Sigma-Aldrich)

7. Standard organic chemistry equipment and supplies: round-bottom flasks, magnetic stirrer, ice bath, apparatus and materials for extraction, drying agent, apparatus for argon atmosphere, Büchner funnel, apparatus, and materials for running and analyzing flash column chromatography, apparatus for rotary evaporation

2.2. Peptide synthesis (see Note 1)

1. 2-Chlorotrityl chloride resin (Sigma-Aldrich)

2. Fmoc-hydrazide (Chem-Impex; Wood Dale, IL, USA)

3. Methanol capping reagent: 5% (v/v) Methanol in dimethylformamide (DMF)

4. Acetyl capping reagent: 5% (v/v) Acetic anhydride in DMF

5. Fmoc deprotection reagent: 20% (v/v) Piperidine in DMF

6. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (EMD Millipore; Burlington, MA, USA)

7. Diisopropylcarbodiimide (DIC)(Sigma-Aldrich)

8. 7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) (Chem-Impex)

9. N-hydroxybenzotriazole (HOBt) (TCI America; Portland, OR, USA)

10. N,N-Diisopropylethylamine (DIPEA) (EMD Millipore)

11. Fmoc-amino acids

12. Fmoc-Glu(Arg(Pdf)-OtBu)-OH (see Subheading 3.1.1.)

13. 4-Pentynoic acid (Sigma-Aldrich)

14. Diazo-biotin-azide (Click chemistry tools; Scottsdale, AZ, USA)

15. Kaiser test reagents: reagent A, 80% (w/v) phenol in ethanol; reagent B, 5% (w/v) ninhydrin in ethanol; and reagent C, 2% (v/v) potassium cyanide in a 1 mM aqueous solution in pyridine.

16. Peptide synthesis apparatus: stir bars, fritted plastic syringes, stoppers, glass vials, tubing, vacuum pump, solvents, mass spectrometry instruments, and reagents for peptide cleavage

17. HPLC buffers: HPLC buffer A consists of Milli-Q water with 0.1% TFA and HPLC buffer B consists of acetonitrile (ACN) with 0.1% TFA.

18. C18 HPLC columns

19. Lyophilizer

2.3. Plasmids and cloning

1. pTXB1-αS-intein-H₆ plasmid: containing α-synuclein (αS) with a C-terminal fusion to the Mycobacterium xenopi GyrA intein and C-terminal His₆ tag. Preparation of this plasmid was described previously[18]. Other inteins can be used, see Note 2.

2. pTXB1-αS-TAG₁₁₄-intein-H₆ plasmid: identical as the above plasmid except the mutation at site 114. Preparation of this plasmid was described previously[19].

3. DNA oligomers for site-directed mutagenesis/deletions

   Primer sequences:

   αS1–76	Forward	5'-TGCATCACGGGAGATGCA-3'
   	Reverse	5'- TGCTGTCACACCCGTCA-3'
   αS91–140-C91	Forward	5'-TGCACTGGCTTTGTCAAAAAG-3'
   	Reverse	5'-CATATGTATATCTCCTTCTTAAAGTTAAAC-3'

4. T100 thermocycler or its equivalent

5. Q5 High-Fidelity 2X Master Mix (New England Biolabs; Ipswich, MA, USA)

6. DNAse/RNAse free water for molecular biology

7. DNA cleanup kit: DNA Clean & Concentrator – 5 (Zymo research; Irvine, CA, USA) or its equivalent

8. Nanoquant plate (TECAN; Zürich, Switzerland) and SPARK (TECAN) plate reader or its equivalent for DNA quantification

9. T4 DNA Ligase (New England Biolabs)

10. T4 Polynucleotide Kinase (PNK) (New England Biolabs)

11. 10x T4 DNA Ligase Buffer (New England Biolabs)

12. DpnI (New England Biolabs)

13. E. coli Dh5α competent cells (New England Biolabs)

14. SOC media (Life Technologies; Carlbad, CA, USA)

15. LB agar plates (Agar 15 g, tryptone 10 g, NaCl 10 g, and yeast extract 5 g per liter of Milli-Q water) sterilized by autoclaving and supplemented with appropriate antibiotics once cooled down enough.

16. LB medium (Tryptone 10 g, NaCl 10 g, and yeast extract 5 g per liter of Milli-Q water:) sterilized by autoclaving.

17. 1000x Ampicillin stock: 100 mg ampicillin dissolved in 1 mL Milli-Q water, stored at −20 °C.

18. DNA miniprep kit: QIAprep Spin Miniprep Kit (50) (Qiagen; Germantown, MD, USA) or its equivalent

19. Standard molecular biology equipment and supplies: centrifuges, dry bath, water bath

2.4. Buffers for protein expression

1. Resuspension buffer: 40 mM Tris, pH 8.3. Dissolve 4.846 g Tris base in 900 mL Milli-Q water. Adjust the pH to 8.3 with 6 M HCl and bring the final volume to 1 L with Milli-Q water. Filter-sterilize the solutions using a 0.22 μm filter. Each time dissolve 1 protease inhibitor tablet (Roche; Basel, Switzerland) into 20 mL of this just prior to use.

2. Equilibration/Wash buffer 1: 50 mM HEPES, pH 7.5. (Dissolve 11.92 g HEPES in 900 ml Milli-Q water. Adjust the pH to 7.5 with 5 M NaOH and bring the final volume to 1 L with Milli-Q water. Filter-sterilize the solutions using a 0.22 μm filter.

3. Wash buffer 2: 50 mM HEPES, 5 mM imidazole, pH 7.5 (Dissolve 11.92 g HEPES and 340.4 mg imidazole in 900 mL Milli-Q water. Adjust the pH to 7.5 with 5 M NaOH/6 M HCl and bring the final volume to 1 L with Milli-Q water. Filter-sterilize the solutions using a 0.22 μm filter.

4. Elution buffer: 50 mM HEPES, 300 mM imidazole, pH 7.5. Dissolve 11.92 g HEPES and 20.42 g imidazole in 900 mL Milli-Q water. Adjust the pH to 7.5 with 6 M HCl and bring the final volume to 1 L with Milli-Q water. Filter-sterilize the solutions using a 0.22 μm filter.

5. Thiazolidine deprotection buffer: 2 M guanidium, 200 mM phosphate, 30mM TCEP, 100 mM methoxyamine, pH 4. Prepare this buffer freshly. Dissolve 1.91 g guanidine hydrochloride, 240 mg sodium phosphate monobasic and 86.00 mg TCEP in 5 mL Milli-Q water. Adjust the pH to 4.0 with 5 M NaOH/6 M HCl and bring the final volume to 10 mL with Milli-Q water.

6. Thiazolidine deprotection dilution buffer: 200 mM phosphate, 30mM TCEP, 100 mM methoxyamine, pH 4. Prepare this buffer freshly. Dissolve 240 mg sodium phosphate monobasic and 86.00 mg TCEP in 5 mL Milli-Q water. Adjust the pH to 4.0 with 5 M NaOH/6 M HCl and bring the final volume to 10 mL with Milli-Q water.

2.5. Protein expression and labeling

1. E. coli BL21-DE3 competent cells (New England Biolabs).

2. pDule-pCNF plasmid (Addgene #85494): encoding for a modified M. janaschii tyrosyl synthetase capable of charging a suppressor tRNA_CUA with propargyltyrosine (PpY or π). For other unnatural amino acids, see per liter of Milli-Q water: 3.

3. SOC media (Life Technologies).

4. LB agar plates. Per 1 liter of Milli-Q water, add 15 g Agar, 10 g tryptone, 10 g NaCl, and 5 g yeast extract. Sterilize by autoclaving and supplement with appropriate antibiotics once cooled down to 37^oC or below.

5. LB medium. Per 1 liter of Milli-Q water, add 10 g Tryptone, 10 g NaCl, and 5 g yeast extract. Sterilize by autoclaving.

6. 10X M9 salts. Per liter of Milli-Q water, add 60 g sodium phosphate dibasic, 30 g potassium phosphate monobasic, 5 g sodium chloride, and 10 g ammonium chloride.

7. Magnesium (II) sulfate stock: 1M Magnesium (II) sulfate. Dissolve 29.98 g solid magnesium sulfate into 250 mL Milli-Q water. Sterile filter through 0.22 μm bottle-top filter.

8. Iron (II) chloride stock: 15 mg/mL Iron (II) chloride. Dissolve 3.75 g solid iron (II) chloride into 250 mL 1 M HCl. Sterile filter through 0.22 μm bottle-top filter.

9. Zinc (II) chloride stock: 15 mg/mL Zinc (II) chloride. Dissolve 3.75 g solid zinc (II) chloride into 250 mL Milli-Q water. Sterile filter through 0.22 μm bottle-top filter.

10. Calcium chloride stock: 0.01 M Calcium chloride. Dissolve 0.275 g solid calcium chloride into 250 mL Milli-Q water. Sterile filter through 0.22 μm bottle-top filter.

11. Yeast extract stock: 10% w/v Yeast extract. Dissolve 25 g yeast extract into 250 mL Milli-Q water. Autoclave on liquid cycle to sterilize. Store at 4 °C.

12. Glucose stock: 40% w/v glucose. Dissolve 100 g glucose (dextrose) into 250 mL Milli-Q water with stirring at room temperature. Sterile filter through 0.22 μm bottle-top filter.

13. M9 minimal media (per 500 mL media: Make 1X M9 salts by adding 450 mL Milli-Q water to 50 mL 10X M9 salts and sterilize by autoclaving. Once cooled down, add appropriate antibiotics, 1 mL magnesium sulfate stock, 0.5 mL ferrous chloride stock, 0.5 mL zinc chloride stock, 5 μL calcium chloride stock, 1 ml yeast extract stock and 6.25 ml glucose stock. For other media, see Note 4.

14. 1000X Ampicillin stock: 100 mg ampicillin dissolved in 1 mL Milli-Q water, stored at −20 °C.

15. 1000X Streptomycin stock: 100 mg streptomycin dissolved in 1 mL Milli-Q water, stored at −20 °C.

16. 1000x IPTG stock: 1M Isopropyl β-d-1-thiogalactopyranoside (IPTG) in Mili-Q water, stored at −20°C.

17. O-Propargyltyrosine (PpY or π): Synthesis of PpY, needed for site-specific click labeling, was previously described [20].

18. Nickel agarose resin (High Density) (GoldBio; St. Louis, MO, USA)

19. Q700 sonicator (QSonica; Newtown, CT, USA) or equivalent

20. β-mercaptoethanol (BME)

21. 2-Mercaptoethanesulfonate (MESNa) (Thermo Fisher Scientific; Waltham, MA, USA)

22. HPLC buffers: HPLC buffer A consists of Milli-Q water with 0.1% TFA and HPLC buffer B consists of acetonitrile (ACN) with 0.1% TFA.

23. C4 HPLC columns

24. Lyophilizer (Labconco)

25. Atto 488-azide (Sigma-Aldrich). Stock solution: 20 mM Atto 488-azide. Dissolve 1 mg into 55.4 μl fresh DMSO. Store at −20°C.

26. Copper (II) sulfate. Stock solution: 80 mM Copper (II) sulfate. Dissolve 12.8 mg into 1 ml Milli-Q water. Store at −20°C.

27. Tris(3- hydroxypropyltriazolylmethyl)amine (THPTA) (Sigma-Aldrich). Stock solution: 50 mM THPTA in Milli-Q water. Dissolve 21.7 mg into 1 ml Milli-Q water. Store at −20°C)

28. Sodium ascorbate. Stock solution: 100 mM sodium ascorbate in degassed MilliQ water. Dissolve 19.8 mg of sodium ascorbate into 1 ml of the degassed Milli-Q water.

29. Apparatus for argon atmosphere (balloons, syringes, needles, rubber stopper, etc.)

30. Standard molecular biology equipment and supplies: spectrophotometer, orbital incubator, ultracentrifuge with fixed-angle rotor, microcentrifuge, standard dialysis tubing, magnetic stirrer, 20 ml disposable plastic columns.

2.6. Native chemical ligation (NCL)

1. NCL buffer pH 3: 6 M Guanidium, 200 mM phosphate, pH 3.0. Dissolve 5.74 g guanidine hydrochloride and 240 mg sodium phosphate monobasic into 5 mL water. Adjust pH as necessary and bring the final volume to 10 mL. Filter-sterilize the solutions using a 0.22 μm filter. See Note 6 on pH.

2. NCL buffer pH 7: 6 M Guanidium, 200 mM phosphate, pH 7.0. Dissolve 5.74 g guanidine hydrochloride and 240 mg sodium phosphate monobasic into 5 mL water. Adjust pH with 5 M NaOH and bring the final volume to 10 mL. Filter-sterilize the solutions using a 0.22 μm filter. See Note 6 on pH.

3. Sodium nitrite. Stock solution: 500 mM NaNO₂ in water. Dissolve 10.3 mg NaNO₂ into 300 μL water. See Note 6 on pH.

4. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma-Aldrich). Stock solution for NCL/MES conversion: 500 mM TCEP, 6 M Guanidium, 200 mM phosphate, pH 7.0. Dissolve 14.3 mg TCEP into 50 μL NCL buffer pH 7. Adjust pH with 5 M NaOH and bring the final volume to 100 μL. See Note 6 on pH.

5. Stock solution for desulfurization: 1 M TCEP, 6 M Guanidium, 200 mM phosphate, pH 7.0. Dissolve 28.6 mg TCEP into 40 μL NCL buffer pH 7. Adjust pH with 5 M NaOH and bring the final volume to 100 μL. See Note 6 on pH.

6. 4-Mercaptophenylacetic acid (MPAA) (Chem-Impex). Stock solution: 500 mM MPAA, 6 M Guanidium, 200 mM phosphate, pH 7.0. Dissolve 8.41 mg MPAA into 60 μL NCL buffer pH 7. Adjust pH with 5 M NaOH and bring the final volume to 100 μL. See Note 6 on pH.

7. 2-Mercaptoethanesulfonate (MESNa) (Thermo Fisher Scientific). Stock solution: 500 mM MES, 6 M Guanidium, 200 mM phosphate, pH 7.0. Dissolve 8.20 mg MESNa into 60 μL NCL buffer pH 7. Adjust pH if necessary and bring the final volume to 100 μL. See Note 6 on pH.

8. Methyl thioglycolate (MTG) (Sigma-Aldrich). Stock dilution: Dilute 4.47 μL to 100 μL with NCL buffer pH 7. See Note 6 on pH.

9. VA-044 (FUJIFILM Wako Chemical USA Corp.; Richmond, VA, USA). Stock solution: 200 mM VA-044 in degassed water. Dissolve 6.47 mg VA-044 into 100 μL degassed water.

10. Reduced glutathione (GSH) (Sigma-Aldrich). Stock solution: 1 M GSH, 6 M Guanidium, 200 mM phosphate, pH 7.0. Dissolve 30.7 mg GSH into 50 μL NCL buffer pH 7. Adjust pH and bring the final volume to 100 μL. See Note 6 on pH.

11. pH indicator paper for pH 6.4–8.0 range

12. Apparatus for argon atmosphere (balloons, syringes, needles, rubber stopper, etc.)

13. Purified synthetic peptide fragments (see Subheading 3.2.3.)

14. Purified, recombinant protein fragments (see Subheading 3.3.2., 3.3.3. and 3.3.4)

15. HPLC buffers: HPLC buffer A consists of Milli-Q water with 0.1% TFA and HPLC buffer B consists of acetonitrile (ACN) with 0.1% TFA.

16. Lyophilizer

3. Methods

3.1. Synthesis of the Fmoc-Glu(Arg(Pdf)-OtBu)-OH

Procedures are briefly summarized (see Figure 2) in a manner that should be accessible to one familiar with standard organic chemistry practices. Characterization of all compounds by mass spectrometry, ¹H NMR, and ¹³C NMR has been reported previously[7].

Figure 2.

Synthesis of Fmoc-Glu(Arg(Pdf)-OtBu)-OH (4).

1. Coupling of Fmoc-Glu-OAll and Arg(Pbf)-OtBu to form Fmoc-Glu(Arg(Pbf)-OtBu)-OAll: Dissolve Fmoc-Glu-OAll 1 (1.77 g, 3.56 mmol) in 15 mL tetrahydrofuran (THF) in a round bottom flask, stir in an ice-salt bath. Add 2 equiv N-methylmorpholine (NMM) and 1 equiv isobutyl chloroformate (IBCF) and stir for 15 min. Add 1 equiv Arg(Pbf)-OtBu 2, stir for 1 h, then warm gradually to room temperature, and let stir overnight. Filter the reaction over a Büchner funnel and wash it 2x with 5% NaHCO3 and then with brine. Dry the extract over MgSO4, filter, and concentrate to an oil under reduced pressure. The Rf of Fmoc-Glu(Arg(Pbf)-OtBu)-OAll 3 on thin layer chromatography (TLC) should be 0.66 in 5:2:1 ethyl acetate: hexanes: methanol + 0.5% acetic acid.

2. Deprotection of allyl group to form Fmoc-Glu(Arg(Pbf)-OtBu)-OH: Redissolve Fmoc-Glu(Arg(Pbf)-OtBu)-OAll 3 from step 1 in 5 mL of dichloromethane (DCM) degassed with argon. Add 1 equiv phenylsilane (PhSiH3) and 2.5% mol tetrakis(triphenylphosphine)palladium(0) catalyst (Pd(PPh3)4) and stir until the solution turns black. Wash the reaction with 2x with 5% NaHCO3, and back extract the aqueous layer with 5 mL DCM. Wash combined organic layers with brine and dry with MgSO4, filter, and evaporate to dryness. Purify by column chromatography on silica (5:2:1 ethyl acetate: hexanes: methanol combined with 0.125% acetic acid). Azeotrope at least four times with toluene in ethyl acetate to remove as much acetic acid as possible to prevent acetylation side reactions during peptide synthesis. Purified product is obtained as an off-white powder: The Rf of Fmoc-Glu(Arg(Pbf)-OtBu)-OH 4 on TLC should be 0.28 in 5:2:1 ethyl acetate: hexanes: methanol + 0.5% acetic acid. See Note 7.

3.2. Peptide synthesis

3.2.1. Peptide synthesis for site-specific E^Arg antibody generation (anti-arginylated αS)

We describe a procedure for synthesizing two arginylated peptides targeting αS E^Arg₄₆ (CVGSKTKE^ArgGVVH, 5) or αS E^Arg₈₃ (CAVAQKTVE^ArgGAG, 6) on the 30 μmol scale using standard Fmoc-based solid phase peptide synthesis strategy. The structure of the peptides can be found in Figure 3. Characterization of the product by MALDI and analytical HPLC can be found in Figure 4A, 4B and are previously published[8]. See Note 8.

Figure 3.

Arginylated peptide sequences/structures

Figure 4.

Characterization of arginylated peptides: (A,B) MALDI-MS and analytical HPLC (gradient, 2%−60% B over 30 min) of product peptide CVGSKTKE^ArgGVVH (5, A) and CAVAQKTVE^ArgGAG (6, B); (C) MALDI-MS of product peptide library XXXXXE^ArgXXXXXC (7); (D-F) MALDI-MS of product peptide pentynoyl-BVGSKTKE^ArgGVVH (8, D), Biotin-diazo-pBVGSKTKEArgGVVH (9, E) and V*AQKTVE^ArgGAGSIAA-acyl hydrazide (10, F; αS₇₇₋₉₀-V*₇₇E^Arg₈₃-NHNH₂).

1. To a fritted plastic syringe, add 20 mg 2-chlorotrityl resin (100–200 mesh, 1.5 mmol/g), a stir bar and v1:1 dichloromethane (DCM): dimethylformamide (DMF). Make sure the resin is submerged. Swell resin by stirring for at least 30 min.

2. Remove the DCM/DMF by vacuum filtration and wash the resin three times with DMF.

3. Dissolve the first Fmoc-amino acid (2 equiv) in DMF at a concentration of 0.15 M. Add N,N-diisopropylethylamine (DIPEA, 4 equiv) to this and preactivate the solution by gentle mixing. Add the solution to the resin and stir for 15 min at room temperature. Remove the mixture from resin by vacuum filtration and wash the resin three times with DMF, three times with DCM, and three times with DMF.

4. Add 5% methanol in DMF to cap unreacted sites on the resin. Gently stir for 10 min. Remove the mixture from resin by vacuum filtration and wash the resin three times with DMF, three times with DCM, and three times with DMF.

5. Remove the Fmoc group by adding 20% piperidine/DMF and stir for 5 min. Collect the solution by pushing air through. Repeat this once. Wash the resin three times with DMF, three times with DCM, and three times with DMF.

6. Perform Kaiser test for positive control. For Kaiser test: to a small glass tube, add one drop of reagent A, reagent B and reagent C and sample resin (at least a few particles). Resuspend well, and heat the glass tube at 100–110 °C for 2–5 min. You should see a color change from yellow to dark blue (positive). If you see yellow (negative), remake the Kaiser test reagents and test again.

7. Quantify the amount of coupled amino acid by the Fmoc content in the deprotection solution using the absorbance at 301 nm (extinction coefficient: 7800 cm⁻¹ M⁻¹; Fmoc quantification).

8. Couple amino acids to elongate the peptide. Monitor completion of couplings by Kaiser test. For standard amino acids, dissolve Fmoc-amino acid (4 equiv) at a concentration of 0.2–0.3 M and HBTU (3.8 equiv) in DMF. Add DIPEA (8 equiv) and pre-activate the amino acid by gentle mixing. Add the activated mixture to the deprotected peptidyl resin and stir for 30 minutes. Perform washes with DMF and DCM between each coupling. Deprotect Fmoc groups by adding 20% piperidine in DMF twice and stirring for 5 min each. For the coupling of Fmoc-Glu(Arg(Pbf)-OtBu)-OH, activate 2 equiv of Fmoc-Glu(Arg(Pbf)-OtBu)-OH in DMF with 1.9 equiv HBTU and 4 equiv DIPEA and then mix with the resin for 1–2 h at 37 °C. Perform Kaiser test after every coupling. Add fresh reagents or extend coupling time if color change is observed.

9. After further elongation, wash the resin with DCM 4 times. Prepare 2 ml cleavage solution comprised of 90% TFA, 5% TIPS and 5% DCM and cool on ice. Pour 0.5 ml of the solution onto the resin on ice. The rest is stored at 4 °C. Stir it for 30 min at room temperature.

10. Filter and collect the solution into a scintillation vial. Repeat this twice (i.e. 1.5 h cleavage in total). Wash the syringe with the remaining cleavage solution and pool into the scintillation vial.

11. Add the cleavage solution dropwise into 10-fold volume of ice-cold diethyl ether in a Falcon tube. Mix this vigorously and allow the substrate to precipitate on dry ice for 5–10 min.

12. Centrifuge the tube for 5 min at 4 °C (4000Xg) and remove the supernatant by decantation. To further get rid of cleavage cocktail, add 10 ml of ice-cold diethyl ether to each pellet, vortex vigorously, allow the substrate to precipitate on dry ice for 10 min, and centrifuge the tubes again. Repeat this once.

13. Remove the supernatant by decantation and dry the crude product overnight in a fume hood.

14. Dissolve the pellet in an acetonitrile-water mixture (final acetonitrile % v/v < 20% to ensure column binding upon HPLC injection) and filter using a chemically compatible system. Purify by reverse phase HPLC over a C18 column, using 0.1% TFA in water/0.1% TFA in acetonitrile as mobile phase.

15. Check the purity by analytical HPLC and confirm identity by mass spectrometry (Figure 4A, 4B). Combine pure fractions, flash-freeze in liquid nitrogen, and lyophilize.

3.2.2. Peptide library synthesis for pan-E^Arg antibody generation

We describe a procedure for synthesizing the arginylated peptide library XXXXXE^ArgXXXXXC (X: mixture of 19 non-cysteine natural amino acids, methionine is replaced with norleucine to prevent oxidation issues) on the 100 μmol scale for generation of antibodies recognizing arginylation regardless of the surrounding sequence. Our method utilizes Fmoc-based solid phase peptide synthesis strategy and an isokinetic mixture reported previously[21, 22]. See Note 9. The general structure of the peptide library can be found in Figure 3. Characterization of the product by MALDI can be found in Figure 4C.

1. Swell 167 mg 2-chlorotrityl resin in v1:1 DCM/DMF by stirring for at least 30 min in a fritted plastic syringe. Couple the first amino acid Fmoc-Cys(Trt)-OH, cap unreacted sites and remove Fmoc group as described in 3.2.1.1. After Fmoc quantification, resuspend resin in 2 ml v1:1 DCM/DMF, adjust resin amount to 100 μmol by volume, and transfer to a glass vessel.

2. Make isokinetic mixture. Weigh 19 natural amino acids (240 equiv total per residue – 2400 equiv in total; norleucine instead of methionine for better stability; Table 1) following the ratio determined previously[21]. Dissolve each amino acid into DMF so the concentration is 0.3 M. Dissolve 32.4g HOBt (2400 equiv) into combined amino acid solution. Split equally to make 10 aliquots. Flash-freeze and store them at −20°C.

3. Perform coupling with isokinetic mixture. To a thawed aliquot, add DIC (240 equiv, 3.716 mL) and activate the cocktail by mixing gently. Pour the solution onto the resin and stir well for an h at room temperature. Perform Kaiser test and washes with DMF/DCM/DMF.

4. Add 20% piperidine and stir well for 5 min. Remove the solution by vacuum filtration. Repeat this once. Perform Kaiser test to confirm deprotection. Perform washes with DMF/DCM/DMF.

5. Repeat 3 and 4 four times. Transfer the resin to a plastic fritted syringe.

6. Couple Fmoc-Glu(Arg(Pbf)OtBu)-OH (2 equiv) and deprotect Fmoc group as described in 3.2.1.1. Transfer the resin back to a glass vessel.

7. Repeat 3 and 4 five times. Transfer the resin to a plastic fritted syringe.

8. Wash the resin with DCM four times. Prepare 4 mL cleavage solution (Reagent K: 82.5% v/v TFA, 5% w/v phenol, 5% v/v water, 5% v/v thioanisole and 2.5% v/v 1.2-ethanedithiol) and cool on ice. Pour 1 ml of the solution onto the resin on ice. The rest is stored at 4 °C. Perform cleavage and precipitation as described in 3.2.1.1. Dry the crude product overnight in a fume hood.

9. Prepare an acetonitrile-water mixture for lyophilization. Dissolve each pellet, flash-freeze the solution and lyophilize. We got 52% yield starting from the first amino acid, based on the average mass. MALDI characterization of the product library is shown in Figure 4C.

Table 1.

Components of isokinetic mixture.

Reagent	Equiv	MW (g/mol)	Weight for 1 mmol scale(g)
Fmoc-l-Ala-OH	8.16	311.33	2.5404528
Fmoc-l-Arg(Pbf)-OH	15.6	648.77	10.120812
Fmoc-l-Asn(Trt)-OH	12.48	596.67	7.4464416
Fmoc-l-Asp(tBu)-OH	8.4	411.45	3.45618
Fmoc-l- Gln(Trt)-OH	12.72	610.7	7.768104
Fmoc-l-Glu(tBu)-OH	8.64	425.47	3.6760608
Fmoc-Gly-OH	6.96	297.31	2.0692776
Fmoc-l-His(Trt)-OH	8.4	619.71	5.205564
Fmoc-l-Ile-OH	41.76	353.41	14.7584016
Fmoc-l-Leu- OH	11.76	353.41	4.1561016
Fmoc-l-Lys(Boc)-OH	14.88	468.54	6.9718752
Fmoc- l-Phe-OH	6	387.43	2.32458
Fmoc-l-Pro-OH	10.32	337.37	3.4816584
Fmoc-l-Ser(tBu)-OH	6.72	383.44	2.5767168
Fmoc-l-Thr(tBu)-OH	11.52	397.46	4.5787392
Fmoc-l-Trp(Boc)-OH	9.12	526.58	4.8024096
Fmoc-l- Tyr(tBu)-OH	9.84	459.53	4.5217752
Fmoc-l-Val-OH	27.12	339.39	9.2042568
Fmoc-l-Nle-OH	9.12	353.41	3.2230992
19 nat AA total	240

3.2.3. Peptide synthesis for crosslinking and pull-down applications

We describe a procedure for synthesizing an arginylated peptide for photocrosslinking and pull-down applications, Biotin-Diazo-pBVGSKTKE^ArgGVVH (B = 4-benzoyl-L-phenylalanine, p = pentynoyl), on the 3 μmol scale (initial peptide synthesis on 30 μmol scale). Our method includes Fmoc-based solid phase peptide synthesis strategy to synthesize pBVGSKTKE^ArgGVVH, followed by a click reaction to conjugate to a PEG linker bearing biotin. The structure of the peptides can be found in Figure 3. Characterization of the product by MALDI can be found in Figure 4D, 4E. See Note 10.

1. Swell 20 mg 2-chlorotrityl resin in v1:1 DCM/DMF by stirring for at least 30 min. Perform the first amino acid coupling and subsequent elongation as described in 3.2.1, until formation of BVGSKTKE^ArgGVVH-resin.

2. Add 4-pentynoic acid (5 equiv) activated by HBTU (4.5 equiv) and DIPEA (10 equiv) in DMF to the resin. Mix for 30 min.

3. Wash the resin 3 times with DMF, and 3 times with DCM.

4. Cleave the peptides from resin by treating with a cleavage cocktail (90% TFA, 5% TIPS, 5% DCM) for 1.5 h. Pool the cleavage solution into cold ether, collect the precipitate and purify it by reverse phase HPLC using a C18 column and 0.1% TFA in water /0.1% TFA in acetonitrile as mobile phase. Confirm identity of pentynoyl BVGSKTKE^ArgGVVH by mass spectrometry (Figure 4D), pool pure fractions, flash-freeze and lyophilize.

5. Dissolve pentyl-BVGSKTKE^ArgGVVH 5 mg (3.1 μmol) in 400 μL 100 mM sodium phosphate buffer, pH 7. Dissolve diazo-biotin-azide 2 equiv (4.4 mg) in 100 μL DMSO and add to the peptide.

6. Mix 50 μL 100 mM CuSO₄ and 50 μL 100 mM THPCA (tris-hydroxypropyltriazolylmethylamine) and add to the above mixture.

7. Add 1 mg sodium ascorbate (1.8 equiv, 5.7 μmol) and incubate at room temperature for 4 h.

8. Purify by reverse phase HPLC over a C18 column. Use 0.1% TFA in water /0.1% TFA in acetonitrile as mobile phase.

9. Confirm identity by mass spectrometry (Figure 4E) and purity by analytical HPLC. Pool pure fractions and lyophilize.

3.2.4. Peptide synthesis for arginylated protein semi-synthesis

We describe a procedure for synthesizing arginylated peptide fragment V*AQKTVE^ArgGAGSIAA-hydrazide (αS_77–90- V*₇₇E^Arg₈₃-hydrazide) for semi-synthesis of arginylated αS (αS-E^Arg₈₃) on the 30 μmol scale using standard Fmoc-based solid phase peptide synthesis strategy (V*: penicillamine). Hydrazide functions as a thioester surrogate[23], while penicillamine does as a valine precursor[24]. The structure of the peptide can be found in Figure 3. Characterization of the product by MALDI can be found in Figure 4F and was published previously[7].

1. Swell 200 mg 2-chlorotrityl resin in v1:1 DMF/DCM for at least 30 min. Wash the resin three times with DMF, three times with DCM, and three times with DMF.

2. Dissolve Fmoc-hydrazine (1 equiv; 25.4 mg) and DIPEA (2 equiv; 34.8 μL) in DMF. Derivatize the resin by reacting overnight at room temperature. Perform washes with DMF/DCM/DMF.

3. Cap unreacted sites with 5% methanol in DMF, stirring for 10 min. Perform washes with DMF/DCM/DMF.

4. Couple the first amino acid using 4 equiv Fmoc-protected amino acid at a concentration of 0.2–0.3 M, HBTU (3.8 equiv) and DIPEA (8 equiv). Add the activated mixture to the resin and stir for 30 min. Perform washes with DMF/DCM/DMF.

5. Cap unreacted sites by stirring with 5% acetic anhydride for 10 min. Wash the resin three times with DMF, three times with DCM, and three times with DMF.

6. Deprotect Fmoc groups by adding 20% piperidine in DMF twice and stirring for 5 min each. Perform Kaiser test for positive control and Fmoc quantification. From here on, prepare coupling reagents based on this quantification.

7. Couple amino acids and remove Fmoc group for subsequent elongation as described in 3.2.1.1. For coupling Fmoc-Pen(Trt)-OH, stir 4 equiv amino acid at a concentration of 0.4 M with 4 equiv 7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), 4 equiv HOBt, and 8 equiv DIPEA at 60 °C for 1 h. Double or triple couple.

8. Cleave the peptide from the resin. Prepare cleavage cocktail (95% TFA, 2.5% TIPS, 2.5% H₂O) and agitate for 1 h at room temperature. Pool the cleavage solution into cold ether, collect the precipitate and purify by reverse phase HPLC over a C4 column. Use 0.1% TFA in water /0.1% TFA in acetonitrile as mobile phase.

9. Confirm identity by mass spectrometry (Figure 4F) and purity by analytical HPLC.

10. Pool pure fractions and lyophilize the product. We got 28% yield, calculated from hydrazine derivatization (step 6).

3.3. Protein expression for arginylated protein semi-synthesisx

3.3.1. Production of plasmids coding αS fragment-intein constructs

We describe a procedure for deletion PCR on a plasmid containing full-length αS to generate protein fragments used for NCL.

1. Perform Deletion PCR. Mix the following together in a PCR tube on ice in this order: 23 μl nuclease-free water, 25 μl Q5 High-Fidelity 2X Master Mix, 1 μl of pTXB1-αS-intein-H₆ plasmid (< 1 μg/ml) and 0.5 μl each of forward/reverse primer (50 μM).

2. Set the tubes in a thermocycler and run the following program:

   Step	Number of cycles	Temperature	Duration
   1	1	98 °C	0:30 min
   2	20	98 °C	0:10 min
   		70 °C (−0.5 °C/cycle)	0:30 min
   		72 °C	4:00 min
   3	15	98 °C	0:10 min
   		60 °C	0:30 min
   		72 °C	4:00 min
   4	1	72 °C	2:00 min
   5	1	4 °C	hold

3. Clean up the PCR reactions using a DNA cleanup kit. Quantify DNA concentration using the nanoquant plate and a plate reader to check the reactions.

4. Circularize the PCR product by phosphorylating 5’ end of each DNA strand and ligating it with the 3’ end, using T4 PNK and T4 DNA ligase, respectively. Mix the following together on ice: 5 μl nuclease-free water, 1 μL 10X T4 DNA ligase buffer, 2 μL purified PCR product, 0.5 μL DpnI, 0.5 μl T4 DNA ligase and 1 μL T4 PNK.

5. Incubate the reaction at 37 °C for 60 min.

6. Cool the reaction on ice for a few minutes and add 50 μL competent Dh5α cells. Incubate for 15 min.

7. Heat shock the cells at 42 °C for 45 sec and keep them back on ice for 2 min.

8. Add 400 μL SOC media and incubate cells for 1 h at 37 °C with 250 rpm shaking.

9. Plate 200 μL cell suspension onto an agar plate supplemented with 0.1 mg/mL ampicillin and incubate overnight at 37 °C.

10. Pick single colonies and grow each in a culture tube with 5 mL LB supplemented with ampicillin. Incubate cells for overnight at 37 °C with 250 rpm shaking.

11. Isolate cells by centrifuging at 4000 g for 20 min and discarding media. Purify DNA using a miniprep kit.

12. Quantify DNA concentration using the nanoquant plate and a plate reader. Submit samples from each colony for sequencing to check identity. Keep the product at −20 °C.

3.3.2. Production of N-terminal protein fragment

We describe a procedure for recombinant expression and purification of N-terminal protein fragment. The protein is fused to a C-terminal intein tag, which allows for transthioesterification upon addition of excess thiol. To prevent hydrolysis of the thioester, pre-chill all the buffers before use. Characterization of the product by MALDI was previously published[7].

1. Transform plasmid containing αS_1–76 fused to a polyhistidine-tagged GyrA intein from Mycobacterium xenopi (Mxe), following the procedure in 3.3.1., into BL21(DE3) competent cells.

2. Pick single colonies to inoculate primary cultures in 5 mL LB media supplemented with 0.1 mg/ml ampicillin. Grow at 37 °C with 250 rpm shaking for 3–6 h until the culture gets cloudy.

3. Prepare LB media for secondary culture by autoclaving and supplementing 0.1 mg/mL ampicillin. Inoculate secondary cultures at 1.5–2%.

4. Incubate secondary culture at 37 °C in a shaker at 250 rpm until optical density (OD) reaches ~0.6. Induce expression of the gene of interest with 1 mM IPTG.

5. Grow the culture in the shaker-incubator at 18 °C overnight.

6. Split culture into bottles and centrifuge the culture at 4000 rpm, 20 min, 4 °C, using GS3 rotor. Prepare resuspension buffer (see 2.4.) and cool on ice.

7. Discard the media and resuspend cell pellets were in the resuspension buffer. Combine resuspended cells into a metal cup.

8. Sonicate cells in the cup in an ice bath (5 min, 1 s ON, 1 s OFF).

9. Centrifuge the resulting lysate at 14,000 rpm, 25 min, 4 °C, using SS34 rotor.

10. Set up a Ni-NTA affinity column to purify αS_1–76 fused to GyrA intein. Equilibrate 3 mL bed volume Ni-NTA resin by flowing through 10 ml equilibration buffer (see 2.4.). Plug the bottom of the column, resuspend with isolated supernatant, and incubate at 4 °C for 1 h. Pre-chill buffers for wash and elution (see 2.4.).

11. Transfer the slurry onto the column and let go the flowthrough. Wash the resin with 15 mL wash buffer 1 and then with 20 ml wash buffer 2. Add 10–12 mL elution buffer and collect eluted protein of interest.

12. Remove excess imidazole by dialyzing into 3l of 20 mM Tris pH 7.5 buffer overnight at 4 °C.

13. Dissolve 200 mM sodium 2-mercaptoethanesulfonate (MESNa) into dialyzed solution and incubate overnight with agitation at 4 °C for transthioesterification.

14. Remove excess MESNa by dialyzing cleaved proteins into 3l of 20 mM Tris, pH 7.5 buffer overnight at 4 °C.

15. Set up a second Ni-NTA column to remove the free intein from the sample. Equilibrate 3ml bed volume Ni-NTA resin as described in 10 and resuspend it with dialyzed, cleaved proteins before incubation at 4 °C for 1 h.

16. Transfer the slurry onto the column and collect the flowthrough, which should include the protein of interest. Elute with 10 ml elution buffer in case transesterification did not work.

17. Purify the protein fragment-thioester by RP-HPLC over a C4 column.

18. Check the identify of fractions by MALDI-MS, combine desired products and lyophilize. We got 24.1 mg per 1 L of E. coli culture.

3.3.3. Production of C-terminal protein fragment (no labeling)

We describe a procedure for recombinant expression and purification of C-terminal protein fragment without labeling. Expression of proteins with N-terminal cysteine in E. coli results in its production with the initiator methionine, which is cleaved in vivo by endogenous methionine aminopeptidase (MAP). The exposed N-terminal cysteine reacts with endogenous aldehydes to form thiazolidine adducts. Our protocol uses methoxyamine for deprotection of thiazolidine adducts. See Note 11.

1. Transform plasmid containing αS_91–140-C₉₁ fused to a polyhistidine-tagged GyrA intein from Mxe into BL21(DE3) competent cells, grow culture and express the gene of interest, following the procedure in 3.3.2.

2. Follow the identical purification procedure up to running the first Ni-NTA affinity column, as in 3.3.2.

3. Elute the protein of interest and cleave off intein by incubation with 200 mM β-mercaptoethanol (βME) on a rotisserie overnight at room temperature.

4. Remove excess imidazole and βME by dialyzing the cleaved protein of interest into 20 mM Tris, pH 8 buffer overnight at 4 °C.

5. Run a second Ni-NTA column as described in 3.3.2. to remove the free intein from the sample.

6. Purify the protein fragment and its thiazolidine adducts by RP-HPLC over a C4 column.

7. Check the identify of fractions by MALDI-MS, combine desired products and lyophilize. We got 12.2 mg of the adducts per 1l of E. coli culture.

8. Prepare buffers for thiazolidine deprotection (see 2.4.). Redissolve the lyophilized peptides into the deprotection buffer and incubate at 37 °C for a few h.

9. Check the reaction hourly by MALDI-MS.

10. Dilute the reaction with thiazolidine deprotection dilution buffer (see 2.4.) so that guanidium concentration is less than 1 M for HPLC injection.

11. Purify the product by RP-HPLC over a C4 column.

12. Check the identify of fractions by MALDI-MS, combine desired products and lyophilize. This reaction typically gives quantitative yield.

3.3.4. Production of fluorescently labeled C-terminal protein fragment

We describe a procedure for recombinant expression and purification of C-terminal protein fragment with a site-specific fluorescent labeling. Unnatural amino acid mutagenesis via amber codon suppression is used to incorporate o-propargyltyrosine (PPY, π) at a desired position and produce C- terminal fragments for site-specific labeling with Atto 488. Labeling with Atto 488-azide via copper-catalyzed azide-alkyne cyclization is performed before thiazolidine deprotection since we found that the labeling condition simultaneously deprotects most thiazolidine derivatives and oxidizes some portion of N-terminal cysteine. The detailed information of this can be found in our published work[7].

1. Co-transform the following plasmids into BL21(DE3) competent cells: i) the plasmid containing αS_91–140-C₉₁TAG₁₁₄ fused to a polyhistidine-tagged GyrA intein from Mxe; ii) pDule-pCNF plasmid. Plate 200–400 μL cell suspension onto an agar plate supplemented with 0.1 mg/mL ampicillin and 0.1 mg/mL streptomycin.

2. Pick single colonies to inoculate primary cultures in 5 mL LB media supplemented with 0.1 mg/mL ampicillin and 0.1 mg/mL streptomycin. Grow at 37 °C with 250 rpm shaking for 3–6 h until the culture gets cloudy.

3. Prepare M9 minimal media as described above by autoclaving and supplementing 0.1 mg/mL ampicillin. Inoculate secondary cultures at 1–2%.

4. Incubate secondary culture at 37 °C in a shaker at 250 rpm until optical density (OD) reaches ~0.6–0.8.

5. Add π (220 mg/L) to the media and incubate for 10 min.

6. Induce expression of the gene of interest with 1 mM IPTG.

7. Grow the culture in the shaker-incubator at 18 °C overnight.

8. Follow the identical purification procedure up to purifying the protein fragment and thiazolidine adducts by HPLC following lyophilization, as in 3.3.3. We got 5.8 mg of this per 1 L of E. coli culture.

9. Prepare for labeling with Atto 488-azide via copper-catalyzed azide-alkyne cyclization. Degas 20 mM Tris pH 8 buffer for at least 10 min. Redissolve the fragment in 20 mM Tris pH 8. Make sodium ascorbate stock solution and thaw other stock solutions.

10. Prepare catalytic mixture consisting of 2 equiv CuSO4, 10 equiv Tris(3- hydroxypropyltriazolylmethyl)amine (THPTA), and 20 equiv sodium ascorbate. Let it sit for 10 min.

11. Add the catalytic mixture to the protein along with 2 equiv fluorophore.

12. Check product formation by MALDI-MS.

13. Purify labeled fragments by HPLC over a C4 column.

14. Check the identify of fractions by MALDI-MS. Combine desired products or pyruvate-derived thiazolidines and lyophilize.

15. Prepare buffers for thiazolidine deprotection (see 2.4.). Redissolve the lyophilize peptides into the deprotection buffer and incubate at 37 °C for a few h.

16. Check reaction progress, purify and lyophilize as in 3.3.3. The yield from 8 we got was 59%.

3.4. Native chemical ligation for arginylated protein semi-synthesis

We describe a procedure for 3-part ligation to synthesize αS with glutamate arginylation at site 83, unlabeled (Figure 5) or with an orthogonally installed fluorophore (Figure 6). Reaction traces by analytical HPLC and characterization of the purified products by MALDI and/or analytical HPLC can be found in Figure 7. Further characterization of crude reaction can be found in our published work[7].

Figure 5.

Semi-synthesis of αS-E^Arg₈₃

Figure 6.

Semi-synthesis of αS-E^Arg₈₃π⁴⁸⁸₁₁₄ (with fluorescent labeling)

Figure 7. Synthesis of αS-E^Arg₈₃ and αS-E^Arg₈₃π⁴⁸⁸₁₁₄ :

(A) MALDI-MS of product αS_1–90-V*₇₇E^Arg₈₃-NHNH₂ (6) resulting from NCL between αS_1–76-MES (11) and αS_77–90-V*₇₇E^Arg₈₃-NHNH₂ (10); (B) MALDI-MS of conversion of intermediate 12 to αS_1–90-V*₇₇E^Arg₈₃-MES (13); (C) analytical HPLC (gradient, 10–50% B over 30 min) and MALDI-MS of MTG-mediated NCL between αS_1–90-V*₇₇E^Arg₈₃-MES (13) and αS_91–140-C₉₁π₄₈₈¹¹⁴ (19) to give αS- V*⁷⁷E^Arg83C⁹¹π⁴⁸⁸₁₁₄ (20); (D) analytical HPLC and MALDI-MS of in situ desulfurized product αS-E^Arg₈₃π⁴⁸⁸₁₁₄ (21); (E) analytical HPLC (gradient, 10–50% B over 30 min) of MTG-mediated NCL between αS_1–90-V*₇₇E^Arg₈₃-MES (13) and αS_91–140-C₉₁ (15) to give αS- V*⁷⁷E^Arg83C⁹¹ (16); (F) MALDI-MS of in situ desulfurized product αS-E^Arg₈₃ (17).

3.4.1. NCL 1: ligation between αS_1–76-thioester and αS_77–90- V*₇₇E^Arg₈₃-hydrazide

1. Degas pH 7 NCL buffer. Re-dissolve αS_1–76-MES thioester (7.2 mg; 789 nmol; 1 equiv) and αS_77–90- V*₇₇E^Arg₈₃-hydrazide (5.8 mg; 3.16 μmol; 4 equiv) in NCL buffer pH 7 (see 2.6.) to final concentration of 1–3 mM. Prepare stock solution of MPAA and TCEP (see 2.6.). See Note 6 on pH. See Note 12 on equivalents.

2. Add MPAA to 100 mM and TCEP to 40–50 mM final concentrations. Adjust reaction pH to 6.8–7.0.

3. Incubate the reaction at 37 °C with agitation at 500 rpm for overnight. Monitor the reaction by MALDI-MS, and supplement TCEP as necessary.

4. Reduce the reaction with a few μL of the TCEP stock, let it sit at room temperature for ~ 10 min before purification by RP-HPLC over a C4 column.

5. Check the identify of fractions by MALDI-MS, combine desired products and lyophilize. We got 61% yield from this NCL. MALDI characterization of the product can be found in Figure 7A.

3.4.2. NCL 2: ligation between αS_1–90-V*₇₇E^Arg₈₃-hydrazide and αS_91–140-C₉₁/ αS_91–140-C₉₁π⁴⁸⁸₁₁₄ followed by desulfurization, using MPAA as a thiol additive

The second ligation involves thioesterification of αS_1–90- V*₇₇E^Arg₈₃-hydrazide, ligation of the resulting thioester with αS_91–140-C₉₁/ αS_91–140-C₉₁π⁴⁸⁸₁₁₄ and desulfurization of the ligation product αS- V*₇₇ E^Arg₈₃C₉₁ / αS_91–140- V*₇₇ E^Arg₈₃C₉₁π⁴⁸⁸₁₁₄ (i.e. conversion of cysteines and penicillamines used in ligation to the respective native alanines and valines). We describe two strategies for this: here, the use of 4-Mercaptophenylacetic acid (MPAA) as a thiol additive, which allows thioesterification and NCL without purifying the intermediate thioester, thanks to its quenching ability of excess sodium nitrite. The ligation product needs to be purified before desulfurization reaction due to radical quenching ability of MPAA[25]; in 3.4.3, the use of methyl thioglycolate (MTG) as a thiol additive, which allows NCL and desulfurization reaction in a one-pot manner, due to its poor ability of radical quenching[26]. As MTG thioester is not very stable and MTG is not potent in quenching oxidation agents, the acyl hydrazide starting material is converted to a MES thioester and purified. MES thioester goes through in situ conversion to MTG thioester once NCL is set up. Since the latter strategy only requires HPLC purification of full-length protein once, it is potent especially when the NCL yield is low.

This section describes NCL 2 using MPAA as a thiol additive. An alternative procedure using MTG as a thiol additive is described in section 3.4.3 below.

1. Dissolve the product of NCL1, or the peptide acyl-hydrazide (111 nmol) in pH 3 NCL buffer (see 2.6.) for a final concentration of 2–3 mM and chill to −15 °C in an ice-salt bath. Make NaNO₂ stock solution (see 2.6.). See Note 6 on pH. See Note 12 on equivalents.

2. For hydrazide to azide conversion, add 10 equiv NaNO₂ and agitate by magnetic stirring for 15 min at −15 °C. Degas pH 7 NCL buffer and make MPAA and TCEP stock solution (see 2.6.).

3. Add 40–50 equiv MPAA to the mixture, then the partner peptide (150 nmol). Warm the reaction to room temperature and adjust the pH to 6.8–7.0.

4. Add TCEP to 40 mM final concentration and incubate the reaction at 37 °C with agitation at 500 rpm for a few h. Monitor product formation by MALDI-MS.

5. Purify the product by RP-HPLC over a C4 column.

6. Check the identify of fractions by MALDI-MS, combine desired products and lyophilize. We got 32% yield over 2 steps.

7. Degas pH 7 NCL buffer and re-dissolve the purified NCL product in pH 7 NCL buffer to final concentration of 0.4–0.8 mM.

8. Make stock solution of glutathione (GSH), TCEP or radical initiator VA-044 (see 2.6.).

9. Mix the NCL product with GSH, TCEP and VA-044 to the final concentration of 100 mM GSH, 250 mM TCEP and 20 mM radical initiator VA-044. Incubate in an argon-purged tube at 37 °C overnight.

10. Monitor product formation by MALDI-MS.

11. Purify the product by RP-HPLC over a C4 column.

12. Check the identify of fractions by MALDI-MS, combine desired products and lyophilize. We got 4.8% yield over 3 steps (unlabeled construct), calculating from the peptide acyl hydrazide.

3.4.3. NCL 2: ligation between αS_1–90-V*₇₇E^Arg₈₃-hydrazide and αS_91–140-C₉₁/ αS_91–140-C₉₁π⁴⁸⁸₁₁₄ followed by desulfurization, using MTG as a thiol additive

1. Dissolve the product of NCL1, or the peptide acyl-hydrazide (5.10 mg; 481 nmol) in pH 3 NCL buffer (see 2.6.) for a final concentration of 2–3 mM and chill to −15 °C in an ice-salt bath. Make NaNO₂, MESNa, and TCEP stock solutions (see 2.6.). See Note 6 on pH. See Note 12 on equivalents.

2. For hydrazide to azide conversion, add 10 equiv NaNO2 and agitate by magnetic stirring for 15 min at −15 °C.

3. Add 100 equiv MESNa to the mixture. After 10 min, warm the reaction to room temperature and adjust the pH to 6.8–7.0.

4. Add TCEP to 40 mM final concentration, check the pH and incubate the reaction at room temperature for 30 min. Monitor product formation by MALDI-MS.

5. Purify the MES thioester product by RP-HPLC over a C4 column.

6. Check the identify of fractions by MALDI-MS, combine desired products and lyophilize. MALDI characterization of this product can be found in Figure 7B.

7. Degas NCL buffer pH 7. Redissolve the purified intermediate, bearing C-terminal MES thioester (Unlabeled - 1.90 mg; 177 nmol, labeled - 2.10 mg; 196 nmol), together with its ligation partner (Unlabeled - 0.85 mg; 152 nmol, labeled - 1.05 mg; 162 nmol) to 2 mM final concentration. Make a stock dilution of MTG and a stock solution of TCEP (see 2.6.).

8. Add 100 equiv MTG and supplement with TCEP to a 40 mM final concentration. Adjust pH to 6.8–7.0.

9. Incubate the reaction at 37 °C with agitation at 500 rpm for a few h. Monitor product formation by MALDI-MS and analytical HPLC (Figure 7C: labeled construct; Figure 4E: unlabeled construct).

10. Prepare for one-pot desulfurization. Dilute NCL reaction mixture to final protein concentration of 0.4–0.8 mM. Prepare stock solution of VA-044, GSH and TCEP (see 2.6.).

11. Mix the NCL reaction mixture with GSH, TCEP and VA-044 to the final concentration of 100 mM GSH, 250 mM TCEP and 20 mM radical initiator VA-044. Incubate in an argon-purged tube at 37 °C overnight.

12. Monitor product formation by MALDI-MS.

13. Purify the product by RP-HPLC over a C4 column.

14. Check the identify of fractions by MALDI-MS, combine desired products and lyophilize. For unlabeled construct, we got 61% yield over 2 steps from MES thioester. For the labeled construct, we got 29% yield over two steps. Characterization of the labeled product by MALDI and/or analytical HPLC can be found in Figure 7D (labeled), Figure 4F (unlabeled).

4. Notes

1. It is important that peptide synthesis reagents are dry. For reagents that need to be stored in a fridge or a freezer, leave the bottles out for at least 30 min before opening them so that they can warm to prevent condensation.

2. Other inteins such as DnaE inteins could alternatively be used[27, 28]. For each protein of interest, it is recommended that expression yield and cleavage efficiency are compared between different intein fusions.

3. As alternative alkyne-bearing unnatural amino acids, pyrrolysine analogues such as N^ε-butynyloxycarbonyllysine[29] or N^ɛ-o-ethynylbenzyloxycarbonyllysine[30] can be incorporated using an orthogonal pyrrolysyl-tRNA synthetase or its mutant.

4. Unnatural amino acid mutagenesis could also be done using autoinducing media or LB media. The temperature for protein expression could be tuned, as well. The choice of conditions should be made based on previous results with the same incorporation machinery or individual yield optimization for each protein of interest.

5. A lot of other thiol additives have been used in NCL [10]. MPAA is the most used due to its affordability and ease of handling, but other aryl thiols such as 4-hydroxythiophenol or 3-mercaptobenzyl sulfonate could be used as alternative. For alkyl thiols, 2,2,2-trifluoroethanethiol (TFET) could be used instead of MTG. MESNa thioesters are primarily used for storage purposes because of the high stability. The choice of additive can be determined by preferred reactivity of the thioester, but could also be affected by availability, handling preferences or the retention time of the NCL components/products.

6. To adjust the pH of stock solutions for NCL or NCL reaction, add small volumes of NaOH at a time, vortex, and check pH. Repeat this until you get ideal pH. It is important to not basify the solution more than necessary. In the case of the NCL reaction, excessively high pH will result in faster hydrolysis of thioester. Also, too much addition of NaOH and compensation with HCl can result in unnecessarily high salt concentration that may affect solubility of reaction components.

7. When working up step 2, the presence of palladium may lead to the formation of an emulsion layer, depending on the scale. Filtering the reaction through Celite helps get rid of palladium and suppress emulsion.

8. Peptide synthesis can be stopped at different points. For short-term stoppage, coupling reactions can be let go overnight or resin can be stored at room temperature after washes following either coupling or capping, but storing peptides with deprotected termini should be avoided. For long-term storage, perform extensive washes with DCM and vacuum for at least 30 sec to dry out resin and keep it in −20 °C. Before resuming the synthesis, swell resin in v1:1 DMF/DCM for at least 30 min and perform washes. On another note, especially for synthesis of longer peptides, keep resin loading up to 40% of the capacity to prevent interchain association which may cause aggregation.

9. Other approaches for randomizing sequence during peptide synthesis include the split-and-pool approach[31], the spot-synthesis approach[32] and the tea-bag approach[33]. Factors to determine an appropriate method for each application would be labor intensity, reproducibility, the preferred size of the library, or the synthesis scale.

10. Biotin or the crosslinker could be attached at a different position of the peptide. Optimization of linker length is necessary to make sure that biomolecules are pulled down and to achieve proximity-sensing.

11. In vivo cleavage of N-terminal residues by MAP or other aminopeptidases is dependent on sequence context[34] and potentially structural conformation, meaning that cleavage does not work as described above all the time. If you have troubles, you could change the ligation site or alternatively encode an N-terminal tag that can be cleaved off during purification process to expose N-terminal cysteine. The alternative method will also bypass thiazolidine deprotection step.

12. Adding excess C-terminal fragment will often speed up ligation and improve the yield, especially when the thioester peptide is prone to circularization or hydrolysis. The excess C-terminal peptide can be recovered after the reaction unless desulfurization is performed without purifying the ligation product.