Link Search Menu Expand Document
The Pan Lab
How to prepare label-free proteomics samples

How to prepare label-free proteomics samples

  • Before processing， prepare the solutions first:

    • UA: 8M urea in 0.1 M tris/HCl (pH 8.5), 1 mL per sample

    • IAA solution: 0.05 M iodoacetamide in UA, prepare 0.1 mL per sample

    • ABC solution: 0.05 M NH4HCO3 in water, prepare 0.25 mL per sample

    • Note: UA and IAA solution must be freshly prepared and used within a day.

  • Processing the sample: ~~~~

    1. Protein pellets were washed with 1 mL chilled (∼-10 °С) acetone followed by centrifugation at 20800×g for 10 min. 

    2. Protein pellets were gently disaggregated in acetone by vertexing during the first acetone addition.

      After centrifugation, acetone was gently removed and discarded, and a fresh

      1 mL of acetone was added. This acetone washing step was repeated three times. 

    3. Protein pellets were air-dried until all acetone evaporated. 

    4. Solubilized in 2 mL of guanidine buffer (6 M Guanidine HCl, 10 mM DTT in Tris CaCl2 buffer (50 mM Tris, 10mM CaCl2, pH 7.6) and incubated at 60 °С for 1 h. With intermittent vertexing to disrupt and dissolve the protein pellet. 

    5. Mix up to 30µl of a protein extract with 200µl of UA (8 M urea (Sigma, U5128) in 0.1 M Tris/HCl pH 8.5) in the filter unit (Microcon YM-30) and centrifuge at 14,000 x g for 15 min.

    6. Add 200µlof UA to the filter unit and centrifuge at 14,000 x g for 15 min

    7. Discard the flow-through form the collection tube.

    8. Add 100 µl IAA solution and mix at 600 rpm in a thermo-mixer for 1 min and incubate without mixing for 20 min

    9. Centrifuge the filter units at 14,000 x g for 10 min.

    10. Add 100 µl of UA to the filter unit and centrifuge at 14,000 x g for 15 min. Repeat this step twice.

    11. Add 100 µl of ABC(0.05M NH4HCO3 in water) to the filter unit and centrifuge at 14,000 x g for 10 min. Repeat this step twice.

    12. Quantify the protein by the BCA kit.

    13. Add 40 µl ABC with trypsin (enzyme to protein ratio 1:100) and mix at 600 rpm in thermo-mixer for 1 min.

    14. Incubate the units in a wet chamber at 37°C for 12 h

    15. Transfer the filter units to new collection tubes.

    16. Centrifuge the filter units at 14,000 x g for 10 min.

    17. Add 40 µl ABC and centrifuge the filter units at 14,000 x g for 10 min.

    18. Desalt by Pierce™ C18 Spin Columns.

    19. Quantify the peptides concentration.

    20. Frozen dry the samples. ~~~~