Acrylamide (ACR) is an electrophilic unsaturated carbonyl derivative that produces neurotoxicity

Acrylamide (ACR) is an electrophilic unsaturated carbonyl derivative that produces neurotoxicity by forming irreversible Michael-type adducts with nucleophilic sulfhydryl thiolate organizations about cysteine residues of neuronal proteins. N27 cells were characterized based on the presumed involvement of CNS dopaminergic damage in ACR neurotoxicity. Shotgun proteomics recognized a total of 15 243 peptides in N27 cells of which 103 unique peptides exhibited ACR-adducted Cys organizations. These peptides were derived from 100 individual proteins and therefore ~0.7% of the N27 cell proteome was adducted. Proteins that contained ACR adducts on multiple peptides included annexin A1 and pleckstrin homology domain-containing family M member 1. Sub-network enrichment analyses indicated that ACR-adducted proteins were involved in processes associated with neuron toxicity diabetes swelling nerve degeneration and atherosclerosis. These results provide detailed info concerning the Ziyuglycoside II ACR-adducted proteome inside a dopaminergic cell collection. The catalog of affected proteins shows the molecular sites of ACR action and the respective roles of these proteins in cellular processes can offer insight into the related neurotoxic mechanism. Ziyuglycoside II ACR treatment cells were harvested using 0.25% Trypsin-Ethylenediaminetetraacetic acid (EDTA) (2 ml; Gibco) for 3 min and trypsin activity was quenched using 1 mg/ml soybean trypsin inhibitor (2 ml; Roche Diagnostics Indianapolis In USA). Cells were pelleted by centrifugation (100 g for 6 min at 4°C) and then gently washed (3x) in ice-cold 1X phosphate-buffered saline (PBS). Cells were then resuspended Ziyuglycoside II in lysis buffer (20 mM Tris-HCl pH 7.7 1 mM EDTA 150 mM NaCl 0.4% Nonidet P-40 and protease inhibitor) and lysed on snow for quarter-hour in the dark. Lysed cells were centrifuged at 13 0 g for 5 minutes at 4°C and proteins were acetone-precipitated (1 part lysis buffer/6 parts acetone). Proteins were reconstituted in Tris buffer (100 mM; pH 8.8) and treated with 10 μl of 5 mM DTT for quarter-hour (55°C). This was followed by incubation with 2 mg iodoacetamide for 1 hour at space temperature in the dark. Approximately 400 μg of protein was trypsin-digested immediately at 37 °C before online desalting and fractionation. 2.2 LC MS/MS and protein / peptide recognition A Ziyuglycoside II nano circulation liquid chromatograph (U3000 Dionex Sunnyvale CA) coupled to an electrospray ion capture mass spectrometer (LTQ-Orbitrap Thermo San Jose CA) was utilized for tandem mass spectrometry peptide recognition experiments. The sample was first loaded onto a pre-column (5mm × 300 μm ID packed with C18 reversed-phase resin 5 100 and washed for 8 moments with aqueous 2% acetonitrile with 0.04% trifluoroacetic acid. The caught peptides were then eluted onto the analytical column (C18 75 μm ID × 15 cm Pepmap 100 Dionex Sunnyvale CA). The 120-minute gradient was programmed as follows: 95% solvent A (2% acetonitrile + 0.1% formic acid) for 8 minutes solvent B (90% acetonitrile + 0.1% formic acid) from 5% to 50% in 90 minutes increasing from 50% to 90% B in 7 minutes then held at 90% for 5 minutes. Re-equilibration was achieved by reducing solvent B from 90% to 5% in 1 minute and holding at 5% B for 10 minutes. The circulation rate for the analytical column was 300 nl/min. Five tandem mass spectra were collected inside a data-dependent manner following each survey scan. The MS scans were acquired in the Orbitrap to obtain accurate peptide mass measurements and the MS/MS scans were acquired in the linear ion capture using 60 second exclusion for previously sampled Ziyuglycoside II peptide peaks. There were 10 fractions that were Ziyuglycoside II investigated in total. Tandem mass spectra were extracted by Xcalibur version 2.0. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Mascot (Matrix Technology London UK; Rabbit Polyclonal to MCPH1. version Mascot) and Sequest (ThermoFinnigan San Jose CA; version 27 rev. 12). Mascot was setup to search the Sprot_20100810 database (selected for < 0.05. Disease pathways were built using all significant manifestation and binding partner networks. Pathogenic processes were mapped onto the protein network to accomplish a more processed interpretation of ACR neurotoxicity through the potential relevance of specific protein adducts. Connectivity scores > 15 (strongly supported network) were used to.