Abstract
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.
Highlights
Sulfation and phosphorylation are post-translational modifications imparting an isobaric 80-Da addition on the side chain of serine, threonine, or tyrosine residues
In the course of preliminary gel electrophoresis experiments on protein standards, we encountered another chemical modification artifact associated with the gel staining protocol
Sulfation Artifact Caused by Silver Staining of Phosphorylated ERK1—We found that proteins extracted from silverstained gels undergo artifactual sulfation that can potentially be mistaken for genuine phosphorylation
Summary
Material and Reagents—Enolase from the yeast Saccharomyces cerevisiae was purchased from Sigma-Aldrich. Proteins were fixed within the polyacrylamide gel by incubating the entire gel in 5% (v/v) acetic acid in a 1:1 (v/v) water:ethanol solution. Staining was performed by incubating the gel in 0.1% (v/v) silver nitrate (AgNO3) in water for 25 min at 4 °C. For Coomassie Blue staining, proteins were fixed after gel separation and stained in a one-step procedure by incubating the entire gel in 0.1% (w/v) Coomassie Brilliant Blue R-250 in a 1:8:11 (v/v/v) acetic acid:methanol:water mixture for 1 h at room temperature. The gel was rinsed three times in a 1:4:5 (v/v/v) acetic acid:methanol:water solution at room temperature to visualize protein bands. Peptides were analyzed in data-dependent mode where for each 1-s survey scan the three most intense precursor ions with intensity above 10,000 counts were selected for MS2 sequencing with a total duty cycle of 2.5 s. The generated unique list of peptide clusters allowed the direct comparison of peptide abundance between samples in different conditions to identify those showing reproducible and statistically meaningful changes in abundance
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