Silver stain removal using H2O2 for enhanced peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Rapid Commun Mass Spectrom. 2002;16(3):160-8. doi: 10.1002/rcm.555.


Hydrogen peroxide solutions are reported for the removal of silver stain from proteins isolated in polyacrylamide gels. Removal of silver stain prior to in-gel digestion is shown to enhance sensitivity and sequence coverage of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) peptide mass maps. The rate of silver removal using H2O2 is influenced by H2O2 concentration and increases with increasing pH. The presence of complexation reagents such as ammonia from mass spectrometry compatible ammonium bicarbonate solutions enhances the efficiency and speed of H2O2-mediated silver removal. H2O2-mediated silver removal using the described procedure does not appear to have any detrimental effects on proteins but is observed to produce a slightly elevated level of methionine oxidization over that usually observed in in-gel tryptic digestion.

MeSH terms

  • Bicarbonates
  • Electrophoresis, Polyacrylamide Gel
  • Hydrogen Peroxide / chemistry*
  • Hydrogen-Ion Concentration
  • Indicators and Reagents
  • Oxidation-Reduction
  • Peptide Mapping / methods*
  • Peptides / chemistry*
  • Plant Extracts / analysis
  • Proteins / chemistry
  • Serum Albumin, Bovine / chemistry
  • Silver Staining / methods*
  • Solutions
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*


  • Bicarbonates
  • Indicators and Reagents
  • Peptides
  • Plant Extracts
  • Proteins
  • Solutions
  • Serum Albumin, Bovine
  • ammonium bicarbonate
  • Hydrogen Peroxide