Proteomic analysis of rat striatal synaptosomes during acrylamide intoxication at a low dose rate

David S. Barber, Stanley Stevens, Richard M. LoPachin

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

We have hypothesized that acrylamide (ACR) intoxication causes cumulative nerve terminal damage by forming adducts with nucleophilic cysteine sulfhydryl groups on critical presynaptic proteins. To determine the cumulative effects of ACR on the cysteine-containing proteome of nerve terminal, we employed cleavable isotope-coded affinity tagging (ICAT) and liquid chromatography-tandem mass spectrometry. ICAT analysis uses a sulfhydryl-specific tag to identify and quantitate cysteine-containing proteins. Synaptosomes were prepared from striatum of ACR-intoxicated rats (21 mg/kg/day × 7, 14, or 21 days) and their age-matched controls. The synaptosomal proteins of each experimental group were labeled with either light (12 C9 - control) or heavy (13C9 - ACR) ICAT reagent. Results show that ACR intoxication caused a progressive reduction in the ICAT labeling of many nerve terminal proteins. A label-free mass spectrometric approach (multidimensional protein identification) was used to show that the observed reductions in ICAT incorporation were not due to general changes in protein abundance and that ACR formed adducts with cysteine residues on peptides which also exhibited reduced ICAT incorporation. The decrease in labeling was temporally correlated to the development of neurological toxicity and confirmed previous findings that cysteine adducts of ACR accumulate as a function of exposure. The accumulation of adduct is consistent with the cumulative neurotoxicity induced by ACR and suggests that cysteine adduct formation is a necessary neuropathogenic step. Furthermore, our analyses identified specific proteins (e.g., v-ATPase, dopamine transporter, N-ethylmaleimide-sensitive factor) that were progressively and significantly adducted by ACR and might, therefore, be neurotoxicologically relevant targets.

Original languageEnglish (US)
Pages (from-to)156-167
Number of pages12
JournalToxicological Sciences
Volume100
Issue number1
DOIs
StatePublished - Nov 2007

Fingerprint

Corpus Striatum
Synaptosomes
Acrylamide
Proteomics
Isotope Labeling
Rats
Isotopes
Cysteine
Proteins
Labeling
N-Ethylmaleimide-Sensitive Proteins
Affinity chromatography
Dopamine Plasma Membrane Transport Proteins
Liquid chromatography
Proteome
Tandem Mass Spectrometry
Affinity Chromatography
Liquid Chromatography
Mass spectrometry
Toxicity

Keywords

  • Adduct formation
  • Isotope-coded affinity tag
  • Nerve terminal
  • Neurotoxicity
  • Proteomic analysis
  • Toxic neuropathy

ASJC Scopus subject areas

  • Toxicology

Cite this

Proteomic analysis of rat striatal synaptosomes during acrylamide intoxication at a low dose rate. / Barber, David S.; Stevens, Stanley; LoPachin, Richard M.

In: Toxicological Sciences, Vol. 100, No. 1, 11.2007, p. 156-167.

Research output: Contribution to journalArticle

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