Measuring Reactive Sulfur Species and Thiol Oxidation States: Challenges and Cautions in Relation to Alkylation-Based Protocols

Péter Nagy, Éva Dóka, Tomoaki Ida, Takaaki Akaike

Research output: Contribution to journalReview articlepeer-review

18 Citations (Scopus)


Significance: Redox biology is gaining ground in research related to human physiology (metabolism, signaling), pathophysiology (cancer, cardiovascular disease, neurodegeneration), and toxicology (radiation-or xenobiotic-induced damage). A major hurdle in advancing redox medicine is the current lack of understanding the mechanisms underpinning the observed detrimental or beneficial in vivo effects. To gain deeper insights into the underlying molecular pathways of redox regulation, we need to appreciate the strengths and limitations of the currently available methods. Recent Advances: Reactive sulfur species (RSS), including cysteine derivatives of peptides and proteins along with small molecules such as hydrogen sulfide or inorganic polysulfides, are major players in redox biology. RSS-mediated regulation of protein functions is a widely studied mechanism in the field, and considerable efforts have been devoted to the development of selective detection methods. Critical Issues: A large number of available methods rely on an alkylation step to freeze the dynamism of consecutive oxidation and reduction events among RSS at a particular time point inside the cell. This process uses the assumption that alkylation blocks all redox events instantaneously. We argue that unfortunately this is often not the case, which could have serious impacts on detected sulfur species speciation and confound experimental results. Future Directions: Novel technologies and prudent optimization of existing methods to accurately characterize the dynamic redox status of the thiol proteome as well as detailed understanding of regulatory and signaling capacities of protein polysulfidation are crucial to open new routes toward therapeutic interventions.

Original languageEnglish
Pages (from-to)1174-1189
Number of pages16
JournalAntioxidants and Redox Signaling
Issue number16
Publication statusPublished - 2020 Dec 1


  • alkylation
  • cysteine oxidation/reduction
  • hydrogen sulfide
  • persulfides
  • polysulfides
  • redox biology


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