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PreprintCited by 39Redox biology and oxidative stressAdvanced Proteomics Techniques and ApplicationsMass Spectrometry Techniques and Applications

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CysQuant: simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry

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Huang, Jingjing, Staes, An, Impens, Francis, Demichev, Vadim, Van Breusegem, Frank, Gevaert, Kris, Willems, Patrick·2023·Redox Biology
DOI 10.1101/2023.07.22.550135PMC PMC10562924

Preprint — published as 10.1016/j.redox.2023.102908

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Abstract

Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function. Accurate quantification of cysteine oxidation is therefore crucial for decoding protein redox regulation. Here, we present CysQuant, a novel approach for simultaneous quantification of cysteine oxidation degrees and protein abundancies. CysQuant involves light/heavy iodoacetamide isotopologues for differential labeling of reduced and reversibly oxidized cysteines analyzed by data-dependent acquisition (DDA) or data-independent acquisition mass spectrometry (DIA-MS). Using plexDIA with in silico predicted spectral libraries, we quantified an average of 18% cysteine oxidation in Arabidopsis thaliana by DIA-MS, including a subset of highly oxidized cysteines forming disulfide bridges in AlphaFold2 predicted structures. Applying CysQuant to Arabidopsis seedlings exposed to excessive light, we successfully quantified the well-established increased reduction of Calvin-Benson cycle enzymes and discovered yet uncharacterized redox-sensitive disulfides in chloroplastic enzymes. Overall, CysQuant is a highly versatile tool for assessing the cysteine modification status that can be widely applied across various mass spectrometry platforms and organisms.

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Metadata

Authors
Huang, J., Staes, A., Impens, F., Demichev, V., Van Breusegem, F., Gevaert, K., & Willems, P.
Affiliations
  • Ghent University (BE)
  • Charité – Universitätsmedizin Berlin (DE)
  • Vlaams Instituut voor Biotechnologie (BE)
Corresponding author
  • Gevaert, Kris
  • Willems, Patrick
ORCID
Venue
Redox Biology
Publisher
openRxiv
Published
2023-07-24
Volume
67
Pages
102908-102908
ISSN
2213-2317 (ISSN-L)
DOI
10.1101/2023.07.22.550135
PMC
PMC10562924
OpenAlex
W4387095593
Cited by
39
References
1
Citations by year
2026: 14 · 2025: 15 · 2024: 9 · 2023: 1
FWCI
3.46
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Redox Biology (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Ghent University Academic Bibliography (Ghent University) (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Bundesministerium für Bildung und Forschung (161L0221)
  • Fonds Wetenschappelijk Onderzoek (1227020N, G007723N, 12T1722N)
  • Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie
  • Vlaamse regering
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
Keywords
cysteine oxidation, protein abundance quantification, data-independent acquisition mass spectrometry, data-dependent acquisition, spectral library, disulfide bonds, protein redox regulation, chloroplast enzymes, Arabidopsis thaliana, light stress
MeSH terms
Cysteine, Oxidation-Reduction, Proteins, Mass Spectrometry, Sulfhydryl Compounds

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