TY - JOUR
T1 - Proteomic analysis of rat striatal synaptosomes during acrylamide intoxication at a low dose rate
AU - Barber, David S.
AU - Stevens, Stanley
AU - LoPachin, Richard M.
PY - 2007/11
Y1 - 2007/11
N2 - 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.
AB - 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.
KW - Adduct formation
KW - Isotope-coded affinity tag
KW - Nerve terminal
KW - Neurotoxicity
KW - Proteomic analysis
KW - Toxic neuropathy
UR - http://www.scopus.com/inward/record.url?scp=35448985440&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=35448985440&partnerID=8YFLogxK
U2 - 10.1093/toxsci/kfm210
DO - 10.1093/toxsci/kfm210
M3 - Article
C2 - 17698512
AN - SCOPUS:35448985440
SN - 1096-6080
VL - 100
SP - 156
EP - 167
JO - Toxicological Sciences
JF - Toxicological Sciences
IS - 1
ER -