Practical Protein Chemistry - A. Darbre 1989

Methods of solid-phase amino acid sequence analysis
Coupling methods
Carboxyl coupling using carbodiimide

The C-terminal carboxyl group of the peptide is activated by a Water-soluble carbodiimide and covalently coupled to aminopolystyrene and aminopropyl Glass (as well as β-APS).

Previously, activation of N-terminally protected Peptides was performed under anhydrous alkaline conditions using TETA resin at 40–50 °C [48]. Currently, unprotected peptides are used, and the activation itself is carried out under milder conditions to avoid side Reactions of the carboxyl groups in protein chains [3, 36, 49, 50, 72, 76]. It was found that when using aminopolystyrene, coupling at pH 3–5 is preferable to Condensation under alkaline conditions. Conversely, Glass-Based Supports are best used under anhydrous conditions [52, 60]. In addition, combined coupling via peptide carboxyl groups and Lys residues has been proposed [54, 55].

12.4.4.1. Advantages, limitations, and coupling Procedure.

Advantages.

1. Carboxyl coupling can be applied to almost all types of peptides, including arginyl- and lysyl-containing fragments of tryptic digests.

2. Condensation under acidic conditions is convenient in many cases, especially when lysyl-containing peptides are insoluble under the alkaline conditions used in the DITC method.

3. If the peptide contains internal Lys residues with an unknown position in the chain, C-terminal coupling of this fragment is preferred.

4. The method can be used for sequencing at the micro-level.

Limitations.

1. For small peptides, condensation occurs with a yield of 40–70% [52]; in the presence of a C-terminal Lys, the yields are low.

2. Peptides containing more than 30 Amino Acids and Proteins are often coupled with yields of up to 80–90%. For example, for bradykinin and the Insulin B-chain, the yields were 70–75% and ~60%, respectively [73]. In the case of proteins, condensation efficiency can reach 90%. However, the better the coupling of proteins to the support, the worse they undergo the Edman Degradation (possibly due to steric hindrance). Therefore, quantitative coupling of the protein via all COOH groups should be avoided. We found that the best results are obtained when the yield is 10–30% of the theoretical value, with preferential coupling via the C-terminal carboxyl. However, for proteins, the best sequencing results were obtained using the DITC method [37].

Procedure 1. Coupling to aminopolystyrene.

Resin washing. To 30–50 mg of aminopolystyrene, 1 mL of buffer No. 2 (Section 12.2.5) is added, and the suspension is stirred until a purple color appears in the solution. The resin is washed with water (2 x 8 mL), DMF (2 x 1 mL), and centrifuged.

Coupling. The peptide solution is dried in vacuo. The sample must be free of salts and organic acids (e.g., acetic, formic). 5–50 nmol of the peptide is dissolved (under nitrogen) in 50 µL of buffer No. 2. An aliquot is taken to check for complete dissolution. The peptide solution is added to the pre-washed resin. The tube containing the peptide is rinsed with 200 µL of DMF, and the washings are added to the reaction mixture. The rinsing of the tube is repeated (100 µL of DMF). A freshly prepared solution of 2 mg of carbodiimide in a mixture of 20 µL of water and 80 µL of DMF is added under nitrogen. The mixture is stirred for 60–120 min at 30 °C (when working with Glx- and Asx-containing peptides, the condensation time is 30–60 min). The reaction mixture is centrifuged; the supernatant is monitored for unbound peptide content. The resin is washed with DMF, methanol, and buffer I (2 x 200 µL).

Blocking of free amino groups. To the peptidyl-polymer, 200 µL of buffer No. 1 is added under nitrogen, followed by 200 µL of solution No. 1 containing PITC (Section 12.2.6). The mixture is gently stirred for 30 min at 30 °C. The resin is washed with DMF (2 x 200 µL), methanol (2 x 200 µL), centrifuged, and dried in vacuo.

Procedure 2. Coupling to aminopropyl glass.

Peptide preparation. 5–50 nmol of the sample is dried in vacuo. It is dissolved in 50–100 µL of anhydrous TFA and kept under nitrogen at room Temperature for 15 min. It is immediately dried in vacuo over KOH pellets (dry for no more than 15 min; longer drying may render the sample insoluble).

Support washing. The support (taken at a ratio of 1 mg/nmol of peptide) is washed with DMF (4 x 2 mL per 50 mg of glass).

Coupling. To the dried peptide, a freshly prepared solution of carbodiimide (2 mg in 200 µL of DMF) is added. The mixture is sonicated for 1 min. Then, 50 mg of aminopropyl glass is added, and the mixture is deaerated. It is kept under nitrogen (with gentle stirring) at 40 °C for 60 min. It is centrifuged. The supernatant is monitored for unbound peptide content. The peptidyl-glass is washed with DMF and methanol (2 x 200 µL). It is centrifuged and dried in vacuo.