How to prepare TMT-labeled proteomics samples

Protein processing steps are same as label free method.

Before labeling:

1. prepare 100mM TEAB (triethyl ammonium bicarbonates): add 500ml of 1M TEAB to 4.5ml of ultrapure water 

2. prepare 5% hydroxylamine: add 50ml of the 50% hydroxylamine to 450 ml of 100 mM TEAB 

Sample labeling

1. Desalted peptides were reconstituted in 0.1% formic acid. And the peptide concentration was determined using BCA kit.
2. The peptides solution was aliquoted, and dried by vacuum centrifugation.
3. Equilibrate the TMTpro Label Reagents to room temperature in the foil pouch.
4. 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.
5. Briefly centrifuge the tube to gather the solution
6. Measure protein digest concentration using BCA kit
7. Add 20 μl of the TMT pro label reagent to each 100 μl sample.
8. Incubate the reaction for 1hr at room temperature
9. Add 5 μl of 5% hydroxylamine to the sample, then incubate for 15 min to quench the reaction
10. Desalt the peptide solution by C18 spin column
11. Speedvac to dry the labeled peptide