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Updated: Jul 19, 2026
Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation
Published on: June 21, 2021
Detection of reversible protein thiol modifications in tissues
Lynette K Rogers1, Barbara L Leinweber, Charles V Smith
1Center for Perinatal Research, Columbus Children's Research Institute, Department of Pediatrics, The Ohio State University, Columbus, OH 43205, USA. rogersl@chi.osu.edu <rogersl@chi.osu.edu>
This study compares reagents for analyzing protein thiol modifications. N-ethylmaleimide (NEM) is more effective and efficient than iodoacetamide (IAM) and iodoacetic acid (IAA) for thiol alkylation, optimizing disulfide detection in biological samples.
Area of Science:
- Biochemistry
- Proteomics
- Analytical Chemistry
Background:
- Protein thiol (PSH) oxidation/reduction is vital in physiological and pathological processes.
- Assessing reagents and sample processing is crucial for accurate analysis of modified cysteinyl residues.
- Existing techniques for protein thiol modification analysis often overlook critical reagent and sample processing assessments.
Purpose of the Study:
- To critically compare the effectiveness of different reagents for protein thiol alkylation and disulfide reduction.
- To optimize sample processing for minimizing ex vivo oxidation.
- To establish a reliable method for quantifying total disulfide content and identifying changes in specific protein bands.
Main Methods:
- Comparative analysis of N-ethylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) for thiol alkylation.
- Assessment of dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) for disulfide reduction.
- Optimization of sample homogenization to minimize ex vivo oxidation using NEM.
- Quantification of disulfide content using NEM for alkylation, DTT for reduction, and mBBr for labeling and fluorimetric detection.
Main Results:
- NEM demonstrated superior efficiency in thiol alkylation, requiring less reagent, time, and lower pH compared to IAM and IAA.
- DTT in MeOH provided better band resolution for disulfide reduction than TCEP.
- Homogenization in aqueous buffers with NEM effectively minimized ex vivo oxidation in tissue samples.
- A robust method for disulfide quantification and identification of changes in protein bands was established.
Conclusions:
- NEM is a highly effective reagent for protein thiol alkylation, offering advantages in efficiency and conditions.
- DTT in methanol is recommended for optimal disulfide reduction and band resolution.
- The developed methodology enables accurate quantification and identification of disulfide modifications in complex biological samples.
- The identified reagents and techniques are applicable to various separation methods, including two-dimensional electrophoresis.
