Cyanogen bromide cleavage

PE-phytolacain R (1 mg, 38 nmol) was treated at 25°C for 24 h in 100 ml of 70% formic acid containing 1 mg of cyanogen bromide, using a ratio of the modified protein to cyanogen bromide of 1:250 (mol/mol). The reaction mixture was diluted with distilled water, then lyophilized.

Cyanogen Bromide Treatment

Photolabeled membranes were adsorbed onto C-18-derivatized silica gel (1 g of gel/100 mg of membrane protein, EM-Science) which had been washed with 100% acetonitrile followed by 0.1% trifluoroacetic acid in water. The silica gel was washed twice with 10 gel volumes of 0.1% trifluoroacetic acid and then washed three times with 10 gel volumes of acetonitrile/trifluoroacetic acid/water (70:0.1:29.9 v/v/v). The silica gel was equilibrated by washing twice with 10 gel volumes of 0.1 M HCl followed by the addition of cyanogen bromide to a final concentration of 50 mg/ml. After overnight incubation at room temperature in the dark, the silica gel was washed twice with 10 gel volumes of 0.1% trifluoroacetic acid to remove the cyanogen bromide. Cyanogen bromide-generated fragments were eluted from the silica gel with acetonitrile/trifluoroacetic acid/water (70:0.1:29.9 v/v/v). Radioactivity in the eluates as well as in the gel was measured during all steps by -emission spectrometry.

Cyanogen Bromide (CNBr) Digestion and Peptide Mapping of the 60 kDa Fragment.

The electroeluted 60 kDa fragment sample (6 nmol) was diluted with 2 M deionized urea and 0.1 M NH4HCO3, pH 7.8 and reconcentrated in a Centricon-30 microconcentrator. After five repetitions of this procedure to remove residual SDS, the resulting solution (280 L) was treated with 20 L of 110 mM DTT, and the sample was incubated for 1 h at 37 C. The solution was cooled to 25 C, 50 L of a 100 mM solution of iodoacetamide was added (90 equiv per cysteine residue), and the mixture was incubated for 1 h and 30 min at 25 C in the dark. Then 40 mg of solid trichloroacetic acid (TCA) was added to a final concentration of 10%, and the suspension was cooled at 0 C for 40 min. The formed suspension was then pelleted using a microcentrifuge, and the supernatant was removed. The protein pellet was washed two times with cold acetone and then redissolved in 50 L of 70% aqueous trifluoroacetic acid (TFA). A CNBr solution was prepared by dissolving 500 mg in 1.5 mL of acetonitrile and 20 L (6 mol, 100 equiv per methionine) of this solution was added to the protein sample in an Eppendorf tube flushed with argon. The reaction mixture was stored in the dark for 24 h at 25 C. The solution was then diluted with 1 mL of water and lyophilized to dryness. The crude digest was fractionated on a Vydac C-4 column (0.46 ? 25 cm) with a gradient of 95% solvent E/5% solvent F (see General Methods) to 35% E/65% F in 90 min at a flow rate of 0.8 mL min-1. After the HPLC fractions were screened by MALDI-TOF MS, the fraction containing the N-terminal peptide was rechromatographed using a shallower gradient of 70% E/30 % F to 55% E/45% F in 60 min at a flow rate of 0.5 mL min-1. Tryptic digestion of the candidate peptide was carried out in a PerSeptive trypsin column (0.5 ? 3 cm, Poroszyme). The column was eluted with buffer containing 0.05 M Tris-HCl (pH 8.2) and 0.05 M CaCl2. The total digestion time in the column was 30 min at a flow rate of 0.01 mL min-1. The tryptic peptides were collected in a C-18 guard column and washed with solvent E until the pH of the eluate was 2.5, and a Vydac C-18 column (0.46 ? 25 cm) was then connected. The column was eluted using a linear gradient of 95% A/5% B to 45% A/55% B in 50 min. Fractions containing individual peptides were collected and analyzed by MALDI-MS (see above).