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Acetonitrile Extraction Of Peptides Post-Digestion

The majority of protocols for in-gel digestion call for acetonitrile extraction of peptides from the gel plug in an effort to increase recovery of peptides (particularly those hydrophobic in nature) from the gel matrix. Subsequent mass spectrometric analysis requires the removal of organic solvent to facilitate loading onto reverse phase material (such as ZipTips or LC columns). As such, peptide solutions are traditionally taken to complete dryness and reconstituted in aqueous solvent. We have compared this approach with direct analysis of peptides from the supernatant (in ammonium bicarbonate) without organic extraction for the analysis of identical gel plugs containing a known amount of a standard protein.

  1. The following protocol was utilized:
  2. Add 50 μL ABC to gel plugs and hold at room temperature (RT) for 10 min.
  3. Purge liquid, add 50 μL ACN and hold at RT for 10 min.
  4. Repeat steps 1 and 2.
  5. Add 30 μL DTT, heat samples to 60°C and incubate for 10 min.
  6. Allow samples to cool for 20 min.
  7. Purge liquid and add 30 μL IA, incubate the samples for 45 min at RT.
  8. Purge liquid and repeat steps 1 and 2 twice.
  9. Add 10 μL of the trypsin solution to rehydrate the gel plugs, leave for 10 min (Note: The trypsin solution is at a concentration of 10 ng/μL for digestion.)
  10. Add 15 μL ABC and incubate the samples for 4 hr at 37°C.
  11. Add 7 μL 3% formic acid to stop the activity of trypsin.
  12. Removed supernatant.
  13. Added 30 μL acetonitrile and left at RT for 10 min.
  14. Combined extracts, dried down using a Speed Vac.
  15. Reconstituted the peptides in 20 μL 0.1 % formic acid.

The volume of peptide solution for both protocols was 20 μL and the entire solution was loaded onto and eluted from ZipTips for MALDI/MS analysis. The figures below show the spectra obtained for glycogen phosphorylase.