Protein processing steps are same as label free method.
prepare 100mM TEAB (triethyl ammonium bicarbonates): add 500ml of 1M TEAB to 4.5ml of ultrapure water
prepare 5% hydroxylamine: add 50ml of the 50% hydroxylamine to 450 ml of 100 mM TEAB
- Desalted peptides were reconstituted in 0.1% formic acid. And the peptide concentration was determined using BCA kit.
- The peptides solution was aliquoted, and dried by vacuum centrifugation.
- Equilibrate the TMTpro Label Reagents to room temperature in the foil pouch.
- Add anhydrous acetonitrile to each vial (0.5mg vial size to 20μl ACN or 5mg to 200μl ACN), then allow the reagent to dissolve for 5 minutes with occasional vertexing.
- Briefly centrifuge the tube to gather the solution
- Measure protein digest concentration using BCA kit
- Add 20 μl of the TMT pro label reagent to each 100 μl sample.
- Incubate the reaction for 1hr at room temperature
- Add 5 μl of 5% hydroxylamine to the sample, then incubate for 15 min to quench the reaction
- Desalt the peptide solution by C18 spin column
- Speedvac to dry the labeled peptide