5.4.1.4. Label peptides with stable isotopes

  1. Add 20 μl of 100 mM triethylammonium bicarbonate to each tube of dried peptides.
  2. Sonicate for 5 min to solubilize peptides.
  3. Add 20 μl of formaldehyde isotopologues to respective samples.
    3.1. 200 mM CH2O (L)-light to the first sample.
    3.2. 200 mM C2H2O (M)- medium to the second sample.
    3.3. 200 mM 13C2H2O – heavy (H) to the third sample).
  4. Add 2 μl of 1 M light ALD solution (sodium cyanoborohydride) to the first (L) sample and second (M) sample.
  5. Add 2 μl of 1 M heavy ALD solution (sodium cyanoborodeuteride) to the third (H) sample.
  6. Sonicate for 5 min.
  7. If required, centrifuge for 1 min at low speed to collect all of the solution at the bottom.
  8. Leave at ambient temperature in the dark for 90 min.
  9. Add 20 μl of 3.0 M NH4Cl.
  10. Leave at ambient temperature in the dark for 10 min.
  11. Add acetic acid until pH < 2.5.