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Updated: Jun 3, 2026
Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue
Published on: August 28, 2021
Proteomic identification of carbonylated proteins in 1,3-dinitrobenzene neurotoxicity
Stephen R Steiner1, Martin A Philbert
1Toxicology Program, School of Public Health, University of Michigan, 1415 Washington Heights, Ann Arbor, MI 48109-2029, USA.
Abstract:
This study demonstrated that 1,3-dinitrobenzene-induced (1,3-DNB) oxidative stress led to the oxidative carbonlyation of specific protein targets in DI TNC1 cells. 1,3-DNB-induced mitochondrial dysfunction, as indicated by loss of tetramethyl rhodamine methyl ester (TMRM) fluorescence, was initially observed at 5h and coincided with peak reactive oxygen species (ROS) production. ROS production was inhibited in cells pre-treated with the mitochondrial permeability transition (MPT) inhibitor, bonkrekic acid (BkA). Pre-incubation with the antioxidant deferoxamine inhibited loss of TMRM fluorescence until 24h after initial exposure to 1,3-DNB. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and subsequent Oxyblot analysis were used to determine if 1,3-DNB exposure led to the formation of protein carbonyls. Exposing DI TNC1 cells to 1,3-DNB led to marked protein carbonylation 45 min following initial exposure. Pre-treatment with deferoxamine or Trolox reduced the intensity of protein carbonylation in DI TNC1 cells exposed to 1mM 1,3-DNB. Tandem MS/MS performed on protein samples isolated from 1,3-DNB-treated cells revealed that specific proteins within the mitochondria, endoplasmic reticulum (ER), and cytosol are targets of protein carbonylation. The results presented in this study are the first to suggest that the molecular mechanism of 1,3-DNB neurotoxicity may occur through selective carbonylation of protein targets found within specific intracellular compartments of susceptible cells.
Insights
1,3-dinitrobenzene (1,3-DNB) causes oxidative stress and protein carbonylation in neuronal cells. This neurotoxicity may involve selective carbonylation of proteins in mitochondria, ER, and cytosol.
Area of Science:
- Neuroscience
- Biochemistry
- Cell Biology
Background:
- 1,3-dinitrobenzene (1,3-DNB) is a known neurotoxin.
- Oxidative stress is implicated in neurodegenerative diseases.
- The specific molecular mechanisms of 1,3-DNB neurotoxicity are not fully understood.
Purpose of the Study:
- To investigate the molecular mechanisms underlying 1,3-dinitrobenzene-induced neurotoxicity.
- To identify specific protein targets of oxidative carbonylation induced by 1,3-DNB.
- To explore the role of mitochondrial dysfunction and reactive oxygen species (ROS) in 1,3-DNB toxicity.
Main Methods:
- Exposure of DI TNC1 cells to 1,3-DNB.
- Measurement of reactive oxygen species (ROS) production and mitochondrial membrane potential (TMRM fluorescence).
- Use of antioxidants (deferoxamine, Trolox) and mitochondrial permeability transition (MPT) inhibitors (bonkrekic acid).
- Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and Oxyblot analysis for protein carbonylation detection.
- Tandem mass spectrometry (MS/MS) for protein identification.
Main Results:
- 1,3-DNB exposure induced oxidative stress, characterized by increased ROS production and mitochondrial dysfunction.
- Protein carbonylation was observed as early as 45 minutes after 1,3-DNB exposure.
- Antioxidants and MPT inhibitors partially protected against 1,3-DNB-induced cellular damage.
- MS/MS analysis identified specific mitochondrial, ER, and cytosolic proteins as targets of carbonylation.
Conclusions:
- 1,3-DNB-induced neurotoxicity involves oxidative stress and selective protein carbonylation.
- Mitochondrial dysfunction and ROS production are key early events in 1,3-DNB toxicity.
- The findings suggest a novel molecular mechanism for 1,3-DNB neurotoxicity involving targeted protein carbonylation within specific cellular compartments.
