SPEAR: A proteomics approach for simultaneous protein expression and redox analysis

Shani Doron, Nardy Lampl, Alon Savidor, Corine Katina, Alexandra Gabashvili, Yishai Levin*, Shilo Rosenwasser*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


Oxidation and reduction of protein cysteinyl thiols serve as molecular switches, which is considered the most central mechanism for redox regulation of biological processes, altering protein structure, biochemical activity, subcellular localization, and binding affinity. Redox proteomics allows global identification of redox-modified cysteine (Cys) sites and quantification of their reversible oxidation/reduction responses, serving as a hypothesis-generating platform to stimulate redox biology mechanistic research. Here, we developed Simultaneous Protein Expression and Redox (SPEAR) analysis, a new redox-proteomics approach based on differential labeling of reversibly oxidized and reduced cysteines with light and heavy isotopic forms of commercially available isotopically-labeled N-ethylmaleimide (NEM). The presented method does not require enrichment for labeled peptides, thus enabling simultaneous quantification of Cys reversible oxidation state and protein abundance. Using SPEAR, we were able to quantify the in-vivo reversible oxidation state of thousands of cysteines across the Arabidopsis proteome under steady-state and oxidative stress conditions. Functional assignment of the identified redox-sensitive proteins demonstrated the widespread effect of oxidative conditions on various cellular functions and highlighted the enrichment of chloroplastic proteins. SPEAR provides a simple, straightforward, and cost-effective means of studying redox proteome dynamics. The presented data provide a global quantitative view of the reversible oxidation of well-known redox-regulated active sites and many novel redox-sensitive sites whose role in plant acclimation to stress conditions remains to be further explored.

Original languageAmerican English
Pages (from-to)366-377
Number of pages12
JournalFree Radical Biology and Medicine
StatePublished - 20 Nov 2021

Bibliographical note

Publisher Copyright:
© 2021 Elsevier Inc.


  • Chloroplasts
  • Cysteine
  • Mass-spectrometry
  • N-ethylmaleimide
  • Plants
  • Redox proteomics
  • Redox regulation


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