A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry.
The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.
Yan
J X
JX
Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College School of Medicine, Heart Science Center, Harefield Hospital, Middlesex, UK. jun.yan@harefield.nthames.nhs.uk
Wait
R
R
Berkelman
T
T
Harry
R A
RA
Westbrook
J A
JA
Wheeler
C H
CH
Dunn
M J
MJ
D000818:Animals; D006801:Humans; D011506:Proteins / Q000032:analysis*; D051381:Rats; D016622:Silver Staining / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D011506:Proteins