About the mechanism of interference of silver staining with peptide mass spectrometry.
The mechanism by which silver staining of proteins in polyacrylamide gels interferes with mass spectrometry of peptides produced by proteolysis has been investigated. It was demonstrated that this interference increases with time between silver staining and gel processing, although the silver image is constant. This suggested an important role of the formaldehyde used in silver staining development in this interference process. Consequently, a formaldehyde-free staining protocol has been devised, using carbohydrazide as the developing agent. This protocol showed much increased peptide coverage and retained the sensitivity of silver staining. These results were however obtained at the expense of an increased background in the stained gels and of a reduced staining homogeneity.
Richert
Sophie
S
Laboratoire de Spectrométrie de Masse Bio-Organique, UMR CNRS 7509, ECPM, Strasbourg, France.
Luche
Sylvie
S
Chevallet
Mireille
M
Van Dorsselaer
Alain
A
Leize-Wagner
Emmanuelle
E
Rabilloud
Thierry
T
D015180:Electrophoresis, Gel, Two-Dimensional; D005557:Formaldehyde / Q000737:chemistry*; D006834:Hydrazines / Q000737:chemistry*; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D016622:Silver Staining / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006834:Hydrazines; D010455:Peptides; D011506:Proteins; D005557:Formaldehyde; C019289:carbohydrazide