A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization- mass spectrometry

TLDR

High‑throughput protein identification is enabled by genomic data and improved analytics, and silver staining remains the most sensitive method for visualizing proteins on 2‑D PAGE, with several protocols developed for better mass spectrometry compatibility. The authors present a commercial kit, Silver Stain PlusOne, that modifies silver staining for improved mass spectrometry compatibility. The method omits the sensitizing reagent, allowing higher protein loading without saturation and facilitating identification and quantitation. The modified protocol yields 2‑D patterns comparable to other silver stains, produces excellent MALDI and ESI‑MS spectra, and is highly compatible with downstream mass spectrometric analysis.

Abstract

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.