Measuring site occupancy: a new perspective on cysteine oxidation

Area of Science:

• Proteomics
• Chemical Biology
• Molecular Biology

Background:

• Site occupancy of protein modifications is crucial for understanding biological roles but is often overlooked.
• Limited analytical tools exist for precise measurement of modification site occupancy.

Purpose of the Study:

• To develop a novel strategy for simultaneous quantitative analysis of cysteine S-nitrosylation (SNO) and S-sulfenylation (SOH) at single-cysteine resolution.
• To determine the relative oxidation occupancy of modification sites and its role in cellular redox response.

Main Methods:

• Utilized high-resolution mass spectrometry combined with differential reduction and iodoTMT(TM) alkylation.
• Employed an in vivo model of mild oxidative stress in Escherichia coli, inducing SNO and SOH under specific growth and treatment conditions.
• Enriched modified peptides using an anti-TMT antibody for accurate quantification.

Main Results:

• Quantified 114 SNO/SOH modified peptides across 90 proteins, identifying relative modification site occupancy.
• Observed that heavily modified cysteines are not always involved in oxidative stress response.
• Found that residues with low modification levels can be significantly affected by mild oxidative imbalance.

Conclusions:

• The developed method is precise and sensitive for detecting and quantifying endogenous oxidative stress on a proteome-wide scale.
• Provides new insights into the role of modification site occupancy in cellular redox response.
• Highlights that low-level oxidative imbalance can have significant biological consequences.