CysQuant: Simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry
Jingjing Huang,
An Staes,
Francis Impens,
Vadim Demichev,
Frank Van Breusegem,
Kris Gevaert,
Patrick Willems
Affiliations
Jingjing Huang
Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052, Ghent, Belgium
An Staes
Department of Biomolecular Medicine, Ghent University, 9052, Ghent, Belgium; Center for Medical Biotechnology, VIB, 9052, Ghent, Belgium; VIB Proteomics Core, 9052, Ghent, Belgium
Francis Impens
Department of Biomolecular Medicine, Ghent University, 9052, Ghent, Belgium; Center for Medical Biotechnology, VIB, 9052, Ghent, Belgium; VIB Proteomics Core, 9052, Ghent, Belgium
Vadim Demichev
Department of Biochemistry, Charité - Universitätsmedizin Berlin, 10117, Berlin, Germany
Frank Van Breusegem
Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052, Ghent, Belgium
Kris Gevaert
Department of Biomolecular Medicine, Ghent University, 9052, Ghent, Belgium; Center for Medical Biotechnology, VIB, 9052, Ghent, Belgium; Corresponding author. Technologiepark 75, 9052, Ghent, Belgium.
Patrick Willems
Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052, Ghent, Belgium; Department of Biomolecular Medicine, Ghent University, 9052, Ghent, Belgium; Center for Medical Biotechnology, VIB, 9052, Ghent, Belgium; Corresponding author. Technologiepark 71, 9052, Ghent, Belgium.
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.
