Identifying the Elusive Dimerization Product Interfering with Methylsulfonato‐Group Labeling of Cysteines in Proteins

Many biomolecular studies start with labeling a protein with a fluorescent label, spin label, or chemical label. The methanethiosulfonate (mts)‐linking group suffers from a hitherto not‐understood side reaction that leads to label‐dimerization instead of the desired linking of the label to the cysteine of the protein. Using electron paramagnetic resonance and mass spectrometry, the side reaction is studied for the MTSL ((1‐oxyl‐2,2,5,5‐tetramethyl‐Δ‐3‐pyrroline‐3‐methyl) methanethiosulfonate) and the (1‐oxyl‐2,2,5,5‐tetramethylpyrrolidin‐3‐yl) methyl methanethiosulfonate label. At 0.1 mM MTSL, substantial dimer formation is observed within the first 5 h. The reaction pathway is elucidated and the structure of the disulfide‐linked asymmetric dimer is suggested. The reaction seems not to involve the nitroxide or a radical reaction, suggesting that this reaction could also occur for other mts‐linked functional or labeling groups.