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Analytical Chem JoVE (Open Access) Citable · DOI

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

DOI: 10.3791/3766-v
What you'll learn
  • ✓ Perform cysTMT labeling to profile cellular thiol redox states under stress
  • ✓ Apply mass spectrometry to identify oxidized cysteine residues in proteomes
  • ✓ Integrate pathogen inoculation with redox proteomics in plant systems
Protocol

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Difficulty
advanced
Total time
~4–5 days (inoculation + incubation + extraction + labeling + MS analysis)
Model organism
Tomato (Solanum lycopersicum); Pseudomonas syringae (pathogen)
Biosafety
BSL-1

Steps

1
Inoculate tomato leaves with Pseudomonas syringae

Apply bacterial pathogen suspension to tomato leaf tissue to induce cellular stress and elevated reactive oxygen species. Allow sufficient incubation time for infection establishment.

▶ 02:07
2
Extract total protein from treated leaf tissue

Harvest infected tomato leaves and perform tissue homogenization and protein extraction using standard lysis protocols to obtain soluble protein lysates.

▶ 03:13
3
Label free thiols with cysTMT reagent

Treat protein samples with cysTMT isotope tags that covalently bind to reduced cysteine residues, enabling quantitative redox state profiling across the proteome.

▶ 04:21
4
Remove excess unreacted cysTMT reagent

Purify labeled samples to eliminate free tag and apply peptide fractionation to reduce sample complexity prior to enrichment.

▶ 06:07
5
Enrich cysTMT-labeled peptides from digest

Selectively isolate labeled peptide species from the total peptide mixture using affinity-based or chromatographic enrichment methods.

▶ 07:44
6
Analyze samples by liquid chromatography mass spectrometry

Perform tandem MS analysis to identify peptide sequences and measure cysTMT reporter ion intensities for quantification of cysteine oxidation.

▶ 08:43
7
Interpret redox proteome profiles and oxidation patterns

Process MS data to map oxidized cysteine sites across the proteome and correlate redox changes with pathogen stress response in tomato.

▶ 10:02
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