This protocol follows Ma et al. (2023, Journal of Proteome Research) and describes the azide functionalization of TMT-labeled peptides using 3-Azido-1-propanamine.
After the labeling reaction, excess reagent is removed via C18-SepPak column purification, and the product is lyophilized using a SpeedVac concentrator.
The resulting azide-functionalized TMT-labeled peptides can be used for downstream bioorthogonal conjugation, such as click chemistry-based enrichment, imaging, or targeted proteomics applications.
| Item | Item article number | Distributor | Vendor |
|---|---|---|---|
| sterile ddH2O | stock room | ||
| 1M sodium citrate | stock room | ||
| 1M CaCl2 | stock room | ||
| Na2HPO4 | S7907-100G | Merck | SigmaAldrich |
| NaH2PO4 | S0751-100G | Merck | SigmaAldrich |
Begin by making the following stock solutions:
1 M CaCl2
| Reagent | Amount | Final concentration |
|---|---|---|
| sterile ddH2O | 50 mL | |
| CaCl2 | 5.55 g | 1 M |
| TOTAL: | 50 mL |
0.1 M Na2HPO4
| Reagent | Amount | Final concentration |
|---|---|---|
| sterile ddH2O | 50 mL | |
| Na2HPO4 | 709.8 mg | 0.1 M |
| TOTAL: | 50 mL |
0.1 M NaH2PO4
| Reagent | Amount | Final concentration |
|---|---|---|
| sterile ddH2O | 50 mL | |
| NaH2PO4 | 599.9 mg | 0.1 M |
| TOTAL: | 50 mL |
Add reagents in the order listed.
Add CaCl2 last.
This is to avoid precipitation of calcium phosphate.
To avoid precipitation of the CA phosphate, the CaCl2 solution is autoclaved separately from the mixture of sodium salts, and the two solutions are mixed aseptically after cooling.
| Reagent | Amount | Final concentration |
|---|---|---|
| sterile ddH2O | 900 mL | |
| Sodium citrate (1 M) | 2 mL | 2 mM |
| NaH2PO4 (0.1 M) | 10 mL | 1 mM |
| Na2HPO4 (0.1 M) | 10 mL | 1 mM |
| CaCl2 (1 M) | 1.5 mL | 1.5 mM |
| sterile ddH2O | 76.5 mL | |
| TOTAL: | 1 Liter |
Autoclaved buffer should be stored in 4°C.