About the mechanism of interference of silver staining with peptide mass spectrometry

TL;DR

This study investigates how silver staining interferes with peptide mass spectrometry and introduces a formaldehyde-free staining method that improves peptide detection but increases background noise.

Contribution

A novel formaldehyde-free silver staining protocol was developed, reducing interference with mass spectrometry and enhancing peptide coverage.

Findings

Interference increases with time after staining

Formaldehyde plays a key role in interference

New protocol improves peptide coverage

Abstract

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.