Artifactual Sulfation of Silver-stained Proteins

• DOI: 10.1074/mcp.m800327-mcp200
• Publication Date: 2008-10-21T00:14:30Z
• Authors: Marlene Gharib; Maria Marcantonio; Sylvia G. Lehmann; Mathieu Courcelles; Sylvain Meloche; Alain Verreault; Pierre Thibault
• Keywords: 0301 basic medicine; Silver Staining; Mitogen-Activated Protein Kinase 3; Sulfates; Molecular Sequence Data; Thiosulfates; Proteins; Saccharomyces cerevisiae; Hydroxylation; 03 medical and health sciences; Bacterial Proteins; Cricetinae; Phosphopyruvate Hydratase; Escherichia coli; Animals; Humans; Amino Acid Sequence; Amino Acids; Phosphorylation; Artifacts; Peptides

Sulfation and phosphorylation are post-translational modifications imparting an isobaric 80-Da addition on the side chain of serine, threonine, or tyrosine residues. These two post-translational modifications are often difficult to distinguish because of their similar MS fragmentation patterns. Targeted MS identification of these modifications in specific proteins commonly relies on their prior separation using gel electrophoresis and silver staining. In the present investigation, we report a potential pitfall in the interpretation of these modifications from silver-stained gels due to artifactual sulfation of serine, threonine, and tyrosine residues by sodium thiosulfate, a commonly used reagent that catalyzes the formation of metallic silver deposits onto proteins. Detailed MS analyses of gel-separated protein standards and Escherichia coli cell extracts indicated that several serine, threonine, and tyrosine residues were sulfated using silver staining protocols but not following Coomassie Blue staining. Sodium thiosulfate was identified as the reagent leading to this unexpected side reaction, and the degree of sulfation was correlated with increasing concentrations of thiosulfate up to 0.02%, which is typically used for silver staining. The significance of this artifact is discussed in the broader context of sulfation and phosphorylation site identification from in vivo and in vitro experiments.