Novel oxidative modifications in redox-active cysteine residues

Area of Science:

• Biochemistry
• Proteomics
• Cell Biology

Background:

• Reactive oxygen species (ROS) target redox-active cysteine residues in proteins.
• Oxidative modifications of cysteine, such as disulfide bonds and various acid derivatives, modulate protein function.
• Understanding novel cysteine modifications is crucial for elucidating cellular signaling pathways.

Purpose of the Study:

• To identify and characterize low-abundant, novel oxidative modifications of cysteine residues in cellular proteins.
• To investigate the mass spectrometry-based detection of these modifications using advanced analytical techniques.
• To explore the formation pathways of identified cysteine derivatives.

Main Methods:

• Purification of cellular GAPDH using 2D-PAGE.
• Mass spectrometry analysis (nano UPLC-ESI-Q-TOF MS/MS) with selective mass screening.
• Utilized MODi and MODmap algorithms for data analysis.
• Preparation and analysis of model compounds to study reaction pathways.

Main Results:

• Identified unexpected mass shifts (-16, -34, +64, +87, +103 Da) at redox-active cysteines in GAPDH, NDP kinase A, peroxiredoxin 6, and mitochondrial proteins.
• Deduced modifications corresponding to cysteine-to-serine conversion, dehydroalanine (DHA), Cys-SO2-SH, and acrylamide adducts of sulfenic/sulfinic acids.
• Demonstrated pathways for DHA and Cys-SO2-SH formation from thiosulfonate intermediates.

Conclusions:

• Novel oxidative modifications, including thiosulfonate, Cys-SO2-SH, DHA, and cysteine-to-serine conversion, occur at redox-active cysteines.
• These findings expand the known repertoire of cysteine oxidation products beyond sulfenic, sulfinic, and sulfonic acids.
• The study provides new insights into the complex redox signaling landscape mediated by cysteine modifications.