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

## TL;DR

This paper identifies a dimerization reaction that interferes with labeling cysteines in proteins using methylsulfonate groups.

## Contribution

The study reveals the structure of a disulfide-linked dimer formed during labeling and clarifies the reaction pathway.

## Key findings

- At 0.1 mM MTSL, significant dimer formation occurs within 5 hours.
- The dimer is disulfide-linked and asymmetric.
- The reaction does not involve radical or nitroxide chemistry.

## Abstract

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.

Too much of a good thing: At too high concentrations of methylsulfoxide‐(spin‐)labels in the labelling mixture, instead of labelling the cysteine of the protein, the label dimerizes with itself. What is the structure of the dimer? After four decades of puzzle, here is the answer.© 2026 WILEY‐VCH GmbH