Development of a method for the reduction of cystine bridges

The automated determination o f cysteine containing peptides would be complicated if there are internal disulfide bonds present. Methods were therefore investigated to establish a suitable reduction step whose product could subsequently be used in the alkylation reaction with minimal sample pretreatment.

The mass spectrometer was operated under the previously described APCI+ conditions using 50% acetonitrile/ 50% water at a flow rate o f 500 pl/min.

3.3,7.1 Adaption o f a FAB method using ammonium hydroxide solution^^

Ala-Cys-S-S-Cys-Ala (1 mg) was treated with n-butanol (200 pi) and 35% ammonium hydroxide solution (2 drops). The resulting mixture was shaken prior to dilution with 50% acetonitrile/ 50% water (1 ml). The mixture was acidified with TFA (1 drop) and a 10 pi loop injection was made.

The resulting spectrum (see Figure 3.19) showed a base peak at m/z 383 corresponding to MH^ o f the oxidised peptide. The reduced peptide showed a peak at m/z 193 (MfT) with an intensity o f only 6.2%. This method appears therefore not to be sufficiently effective for the reduction of cysteine bridges due to the reduced solubility o f the dipeptide in n-butanol compared to water.

Relative Intcnsitv 383 _{^E + 05} 4.29 100 80 60 40 113 405 20 105 365 64 193 234 80 127 159 200 241 266 225 44 iti/'z 100 200 300 400

Figure 3.19 : Full scan spectrum of Ala-Cys-S-S-Cys-Ala treated with n-butanol and ammonium hydroxide

3.3.7.2 Reduction using aqueous dithiothreitol (DTT)^^

Ala-Cys-S-S-Cys-Ala (1 mg) was treated with an aqueous solution o f 50 mM DTT (200 pi) at 50°C for 15 minutes. On cooling, this reaction solution was diluted with 50% acetonitrile/ 50% water (800 pi) and a 10 pi loop injection was made.

The resulting spectrum (see Figure 3.20) showed a base peak at m/z 193 corresponding to M fT o f the reduced peptide. A significant ion (90% relative intensity) was also observed at m/z 234 due to an acetonitrile adduct o f this species. No peak was observed at m/z 383 for the oxidised peptide. This method therefore appears to be suitable for the effective reduction o f cystine bridges.

Relarivc Intensicy

r

Figure 3.20 : Full scan spectrum of Ala-Cys-S-S-Cys-Ala reduced with aqueous DTT

3.3.7.3 Reduction using dithiothreitol (DTT) in n-butanol

The DTT method (see Section 3.3.7.2) was adapted to investigate the use of an alcohol instead o f water in attempts to make the method more compatible with the subsequent alkylation step.

A 50 mM DTT solution was prepared in a 5% water/ 95% n-butanol. Ala-Cys-S-S-Cys- Ala (1 mg) was treated with this solution (200 )li1) at 50°C for 15 minutes. On cooling, a

10 p.1 loop injection was made.

The resulting spectrum (see Figure 3.21) showed a base peak at m/z 383 corresponding to MH^ o f the oxidised peptide. Peak intensities o f only approximately 15% and 16% were observed respectively for the ions of interest at m/z 193 and m/z 234 due to MFT and [MH^ + CH3CN] respectively of the reduced peptide. It would therefore appear that the reduced solubility o f the peptide in n-butanol significantly affects the rate of reaction.

R elative Intensity 383 _{^E + 05} 3.25 100 80 60 40 115 234 20 193 365 100 129 154 266 250 I 279 312 345 405 182 W6 100 200 300 400

Figure 3.21 : Full scan spectrum of Ala-Cys-S-S-Cys-Ala treated with DTT in n-butanol

3.3.7.4 Conclusions o f cystine bridge reduction step

It would appear that the reduction o f the cystine bridges proceeds best in an aqueous solvent. This is probably due to the poor solubility o f the peptide in n-butanol. Future reductions will therefore be carried out in water as described in Section 3.3.7.2. Unfortunately, as water is incompatible with the butylation and pentylation steps, the reaction mixture requires freeze-drying prior to alkylation o f the dipeptides.

.96 3.3.8 S-Carboxyamidation of cysteine containing dipeptides with iodoacetamide

Peptides containing reduced cystine bridges are prone to oxidation, therefore the HS- group requires protection with a stable group. Iodoacetamide (ICH2CONH2) is an alkylating agent commonly used for this purpose.

Ala-Cys-S-S-Cys-Ala (1 mg) was reduced using DTT (200 \x\) as described in Section 3.3.7.2. On cooling, the solution was treated in two separate experiments with 45 mM

iodoacetamide solution was added for 15 minutes and in the second experiment, 100 pi of the iodoacetamide solution was used for 20 minutes. The resulting solutions were freeze- dried prior to being redissolved in 50% acetonitrile/ 50% water (1 ml) and analysed as described above.

Both experiments produced spectra (see Figures 3.22 and 3.23) showing a base peak at m/z 250 corresponding to MH^ o f the acetamide-derivatised peptide. Significant ions (15- 20% relative intensity) were also observed at m/z 291 due to the acetonitrile adduct of this species. Ions were observed at m/z 383 (approximately 10% relative intensity) corresponding to MH^ o f the oxidised peptide. Ions at m/z 193 and m/z 234 corresponding to MH^ and [MH+CHsCN]^ o f the reduced, non-S-carboxyamidated form are significantly weaker in the spectrum from the second experiment than from the first.

Relative Intensity

100 - E +06

Figure 3.22 : Full scan spectrum o f Ala-Cys-S-S-Cys-Ala reduced with aqueous DTT and treated with 50 pi iodoacetamide for 15 minutes

R elative Intensity 100 H 80 - 60 . E+ 06 40 - 20 -

Figure 3.23 : Full scan spectrum o f Ala-Cys-S-S-Cys-Ala reduced with aqueous DTT and treated with 100 pi iodoacetamide for 20 minutes

It would therefore appear that the reaction proceeds to almost completion when the reduced peptide is treated with a greater proportion o f iodoacetamide for a longer period. This method was therefore selected for all future S-carboxyamidations.