Cysteine Promoted C-Terminal Hydrazinolysis of Native Peptides and Proteins

Tagging the terminus: N→S acyl transfer in native peptides and proteins can be reliably intercepted with hydrazine. The method allows selective labeling and ligation, without recourse to the use of protein-splicing elements. NCL=native chemical ligation.


C-NMR studies
Model peptide was dissolved at 1 mg/mL concentration in 0.1 M sodium phosphate buffer (pH 5.8), MESNa (10% v/w) and hydrazine acetate (5% w/v) in D 2 O. This reaction was shaken on an Eppendorf thermomixer at 600 rpm at 40-60 °C. In order to follow hydrazide formation, 0.5 mL aliquots were removed at 0 h, 6 h, 24 h and 48 h for 13 C NMR analysis. Hydrazide was formed in both cases, complete in 24 h for model-OH and in 48 h for model-NH 2 .

Synthesis of internal Gly-Cys 13 C-labeled model peptide:
Peptide H-MEELG( 13 C-1)CYKS-OH was manually prepared using preloaded Fmoc-Ser(Trt)-NovaSyn TGT resin , loading = 0.24 mmol/g (208 mg, 0.05 mmol). The resin was placed in a sintered filter tube and treated with 20% Fractions containing the deprotected peptide were collected from the HPLC and diluted using 40% acetonitrile/water to give an estimated concentration of 0.16 mg/mL of peptide. The pH was adjusted to pH 7.5 using 30% ammonium hydroxide solution and 3 equivalents each of oxidized and reduced glutathione were added.
The reaction was shaken gently overnight at room temperature, then neat trifluoroacetic acid was added to lower the pH to pH 2. The solution was lyophilized, redissolved in 30% acetonitrile/water and purified by rp-HPLC.

Expression of ubiquitin:
The WT ubiquitin clone was supplied by Dr L. Cabrita and Dr J. Christodoulou in a pET2b(+) plasmid.
The ubiquitin G76C mutant was cloned from the WT ubiquitin pET2b(+) plasmid into a pNIC28-Bsa4 vector with TATCCACCTTTACTGTCAACAACCACGTAGACGTAAGAC as the forward and TATCCACCTTTACTGTTAACAACCACGTAGACGCAAGAC reverse primers respectively. DNA sequencing confirmed the identity of the construct and the vector was heat-shock transformed into E. coli The protein eluted with one peak of a mass of approximately 12 kDa corresponding to a monomer.
Fractions containing the protein were pooled, exchanged into a pH 5.8 buffer (100 mM sodium phosphate buffer pH 5.8, 100 mM NaCl), using Amicon Ultra devices (Millipore) and concentrated to 1 mg mL -1 . The purity and identity of the protein was confirmed by mass spectrometry, with a yield of approximately 6 mg L -1 of cell culture.

Hydrazinolysis of recombinant ubiquitin sample
Purified ubiquitin was obtained at 1-2 mg/mL concentration, in 0.1 M NaCl, 0.1 M Na Phosphate buffer solution (pH 5.8, 0.9 mL) by use of a cerntifugal filter equipped with a 3KDa molecular weight cut off. 2-Mercaptoethanesulfonate (100 mg) was added followed by tris (2-  ii) with cysteamine (10). Ubiquitin C-terminal hydrazide (obtained as above), dissolved at 1-2 mg/mL concentration, in ligation buffer (pH 4, 0.25 mL) was cooled to 0 o C with gentle magnetic stirring. Sodium  Proteo 90A, C 12 , 250 x 21.2 mm column, using a mobile phase of 0.1% TFA (v/v) in water /acetonitrile over a 5-60% acetonitrile gradient. Fractions contianing the protein fragment were identified by LC-MS and lyophilized to obtain the product as a fluffy white solid.

LC-MS after 1h reaction
Hydrazinolysis was then carried out by dissolving the protein in 6M guanidine hydrochloride containing 1% TCEP hydrochloride. MESNa was then added to a final concentration of 10% w/v, followed by hydrazine acetate (5% w/v) and the reaction pH adjusted to pH 5 if necessary. Reactions were incubated at 45 o C for 48 hours, followed by buffer-exchange into 6M guanidine using a 3000 MWCO centrifugal concentrator. Reactions were replenished with fresh reagents, and resumed for a further 48 hours.
Note 1: Alternative solubilization buffer systems to 6 M guanidine.HCl, such as 6 M urea, 1 % sodium dodecyl sulfate, n-ocyl maltoside, and 2 % N-lauroyl sarcosine were also examined for the solubilisation and hydrazinolysis of EPO without success.
Note 2: For synthesis purposes hydrazinium acetate can be replaced with hydrazine hydrate or hydrazine dihydrochloride, but the pH of stock solutions of these reagents must first be adjusted (with