Evidence that the ProPerDP method is inadequate for protein persulfidation detection due to lack of specificity

Abstract

Protein persulfidation (protein-SSH) is a previously unidentified type of modification found in both eukaryotic and prokaryotic cells in recent years. Although a few persulfidated proteins have been identified, analyzing protein persulfidation from a proteomic level is still a big challenge. ProPerDP is a persulfidation detection method recently reported in Science Advances. The authors claimed that this method could specifically detect persulfidated proteins of cell lysate with minor false-positive hits; hence, it could be used for proteomic-level analysis of protein persulfidation. However, when using this method for Escherichia coli cell lysate analysis, we found that the percentage of false-positive hit was >90%. We performed a systematic study on this method and discovered that iodoacetyl-PEG2-biotin tag mislabeling is the reason causing this low specificity. We concluded that the ProPerDP method is completely inadequate for persulfidation analysis. The previous findings based on the ProPerDP method need to be reinvestigated.

Comment on “Evidence that the ProPerDP method is inadequate for protein persulfidation detection due to lack of specificity”

“…Therefore, contrary to the conclusions by Fan et al . ( 5 ), there is nothing unexpected in their finding that only a low percentage of the peptides they analyzed contained Cys.…”

“…From the experimental information provided, it remains unclear whether Fan et al . ( 5 ) validated their deviation from the established protocol with a known system before applying these conditions to the E. coli system in their paper or not.…”

“…Fan et al . ( 5 ) studied the effects of deletion of OxyR, a redox-regulated transcription factor, on the global persulfidation levels in Escherichia coli . They had previously found that OxyR deletion promoted accumulation of total polysulfide levels in E. coli ( 6 ) and hence expected protein persulfidation to increase concomitantly in the mutant strain.…”

“…Nonetheless, we also note that the list of proteins reported by Fan et al . ( 5 ) that were detected as persulfidated and identified by liquid chromatography–tandem mass spectrometry [data file S1 in ( 5 )] revealed that 36 of the 132 proteins (27%) lack Cys residues in their native sequence, implying that there was at least some nonspecific capture on the streptavidin matrix they used. In their paper, Fan et al .…”

“…In their paper, Fan et al . ( 5 ) used streptavidin agarose resin for the affinity pull-down, whereas the original ProPerDP method uses magnetic streptavidin beads ( 8 ). A technical report on how to perform immunoprecipitation suggests using magnetic beads rather than agarose resin for protein separations ( 9 ).…”