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Supplementary Information for Proteome Analysis Supplementary Information Targeting redox metabolism: the perfect storm induced by acrylamide poisoning in the brain Demetrio Raldúa, Marta Casado, Eva Prats, Melissa Faria, Francesc Puig-Castellví, Yolanda Pérez, Ignacio Alfonso, Chuan-Yu Hsu, Mark A. Arick II, Natàlia Garcia-Reyero, Tamar Ziv, Shani Ben-Lulu, Arie Admon, Benjamin Piña Supplementary information for proteome analysis Proteomic Analysis Protein fraction were extracted from 5 pools (of 3 brains each) from control group and 5 pools (of 3 brains each) from AA-treated adult zebrafish. Proteins were trypsinized and 2ug of tryptic peptides from each samples analyzed by LC-MS/MS using a Q- Exactive-Plus mass spectrometer fitted with a capillary HPLC. Proteomic data Analysis The mass spectrometry data were analyzed using the MaxQuant software 1.5.2.8 (www.maxquant.org) ((Cox & Mann, 2008) fitted with the Andromeda (Cox et al., 2011) search engine searching against the Danio rerio Uniprot database (of March 2017 containing 59,064 entries) with mass tolerance of 20 ppm for the precursor masses and the fragment ions. Oxidation on methionine, propionamide on cysteine, histidine and lysine, and carbamidomethyl on cysteine was accepted as variable modifications. Minimal peptide length was set to six amino acids and a maximum of two miscleavages was allowed. Peptide and protein level false discovery rates (FDRs) were filtered to 1% using the target-decoy strategy. The identified protein table was filtered to remove the identifications from the reverse database, the common contaminants and single peptide identifications. Data were quantified by normalized label free analysis using the same MaxQuant software (LFQ intensities), based on extracted ion currents (XICs) of peptides enabling quantitation from each LC/MS run for each peptide identified in any experiment. Ratio mod/base was calculated for each samples by the MaxQuant software. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized ppb-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367 (2008). Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011). Supplementary Figure SF1 Supplementary Figure SF1. (A) Partial 1H NMR spectra of zebrafish brain extract (green trace) and treated with AA (black trace) shoWing diagnostic signals for the identification of the reduced and oxidized forms of glutathione (signal assignment in the upper chemical structures). (B) J-RES NMR spectra for the corresponding untreated (green) and AA-treated (black) of zebrafish brain extracts. (C) Partial 1H NMR spectra of zebrafish brain extract (green trace) and treated With AA (black trace) shoWing diagnostic signals for the identification of AAMA and MetSO. (D) Spiking experiments for the confirmation of the presence of AAMA. (E) Spiking experiments for the confirmation of the presence of MetSO. Supplementary Figure SF2 Supplementary Figure SF2. 1H-1H COSY experiment shoWing representative signals of oxidized glutathione (GSSG) observed in zebrafish brain extracts. Relevant GSSG resonances are indicated by loW-case letters, Which correspond to the positions indicated in the molecular formula represented on the top of the figure. Supplementary Figure SF3 Supplementary Figure SF3. 1H-1H COSY (up) and 1H-13C HSQC (doWn) experiments to confirm AAMA assignment. The graphs show results from a ACR-treated zebrafish brain extract spiked With 0.5mM AAMA. Relevant AAMA resonances are indicated by loW-case letters, Which correspond to the positions indicated in the molecular formula represented on the top of the figure. Supplementary Table ST1. Proton, carbon and J-coupling values for low molecular weight brain zebrafish metabolites. Chemical shifts are reported with reference to DSS (at 0.00 ppm) and multiplicity definitions are s (singlet), d (doublet), t (triplet), dd (doublet of doublets) and m (multiplet). 1H-1H Confirmed in Confirmed in Confirmed in observed # Metabolite Group 1 Peak intensity/area 13 J (in Hz) Notes H/ppm C/ppm 1 HH correlations COSY/TOCSY? HSQC? JRES ? H multiplicity 1 N-Acetyl-S-(carbamoylethyl)-L-cysteine Derived form adduct between GSH and AAM. Confirmed by spiking 1 - CH 4.36 tiny n.o. no n.o. no no n.o. n.m. Partially overlapped with N-Acetyl-L-Aspartic Ac. 2 1H-CH2 3.06 tiny n.o. no n.o. no yes dd 13.66, 4.32 Partially overlapped with Creatine and Carnosine 2 1H-CH2 2.89 tiny n.o. no n.o. no yes dd 13.61, 8.00 Partially overlapped with DSS 3 -CH2 2.82 small yes n.o. no yes dd 6.99 One of the J's is very small; overlapped with L-Aspartic Ac. in treated brain samples 2.58-2.82 (COSY) 4 -CH2 2.58 small yes n.o. no yes dd 7.00 One of the J's is very small 5 -CH3 2.04 small n.o. no yes s 2 Acrylamide CH 5.81 tiny 6.22-5.81 yes n.o. no yes dd 10.01, 1.47 1H-CH2 6.22 tiny 6.22-5.81 yes n.o. no no n.o. n.m. 1H-CH2 6.28 tiny n.o. no n.o. no no n.o. n.m. 3 ATP 6.14-4.79 (TOCSY) 1 - CH 6,14 medium 6.14-4.59 (TOCSY) yes 6.14-89.26 yes yes d 5.78 Anomeric sugar protons from nucleosides: ATP+AMP+Inosinic Acid 6.14-4.39 (TOCSY) 2 -CH 4.80* n.o. 6.13-4.80 (COSY) yes n.o. no no n.o. Below water supressed resonance 3 -CH 4.39 tiny 4.59-4.39 (TOCSY) yes n.o. no yes m 4 -CH 4.59 tiny 4.59-4.39 (TOCSY) yes n.o. no no n.o. 5 -CH2 4.21-4.28 tiny 4.39-4.21 (TOCSY) yes n.o. no no n.o. 6 -CH 8.52*** medium n.o. no n.o. no yes s 7 -CH 8,26 medium n.o. no 8.25-155.65 yes yes s Overlapped with AMP 4 AMP 6.13-4.50 (TOCSY) 1 - CH 6,13 small yes 6.14-89.26 yes yes d 5.85 6.13-4.80 (TOCSY) 2 -CH 4.80* n.o. 6.13-4.80 (COSY) yes n.o. no no n.o. Below water supressed resonance 3 -CH 4.50 small 4.50-4.36 (TOCSY) yes n.o. no yes m 4 -CH 4.36 small 4.36-4.50 (TOCSY) yes n.o. no no n.o. Overlapped with N-acetyl-L-aspartic acid and Inosinic acid 5 -CH2 4.02* n.o. n.o. no n.o. no no n.o. 6 -CH 8.58*** medium n.o. no n.o. no yes s Overlapped with Inosinic Acid 7 -CH 8.25 medium n.o. no 8.25-155.65 yes yes s Overlapped with ATP 5 Betaine - (CH3)3N+ 3.25 medium 3.25-3.89 (COSY) yes 3.25-56.15 yes yes s Overlapped with D-glucose - CH2 3.89 small 3.89-69.03 s Overlapped with L-Aspartic Acid 6 Carnosine 1 -CH 8.22 large yes 8.22-136.85 yes yes d 2.40 8.22-7.12 (COSY) 2 -CH 7.12 large yes 7.12-119.83 yes yes d 2.72 3 1H-CH2 3.19 medium 3.19-3.02 (TOCSY) yes 3.19-30.83 yes yes dd 15.10, 5.00 Overlapped with Choline/Phosphorylcholine 3 1H-CH2 3.02 medium 3.02-3.19 (TOCSY) yes 3.02-30.83 yes yes dd 15.20, 8.50 Overlapped with GABA and Creatine 4.45-3.18 (COSY) 4 -CH 4.45 medium yes 4.45-57.24 yes yes dd 8.40, 4.80 4.45-3.02 (COSY) 5 -CH2 3.22* n.m. n.o. no n.o. no m m n.m. Overlapped with GPC 6 -CH2 2.68* n.m. n.o. no n.o. no m m n.m. Overlapped with N-Acetyl-L-Aspartic Ac. and L-Aspartic Ac. 7 Choline+PC+ GPC (Choline moiety) 1 -CH2 4.06 tiny n.o. no n.o. no no n.o. n.m. Tiny resonance, overlapped with myo-inositol 2 -CH2 3.52* n.o. n.o. no n.o. no no n.o. n.m. Tiny resonance, overlapped with myo-inositol 3.22 large n.o. no 3.22-56.72 yes yes s Assigned to Glycerophosphocholine; Overlapped with Carnosine 3,4,5 -(CH3)3N+ 3.19 small n.o. no 3.19-56.64 yes yes s Assigned to Choline/Phosphorylcholine GPC Glycerol moeity 1 -CH2 3.94/3.88 tiny n.o. no 3.94/3.88-69.27 yes no n.o. n.m. Overlapped with Creatine/Betaine 2 -CH 3.91 tiny n.o. no 3.91-73.31 yes no n.o. n.m. Overlapped with Creatine/Betaine 3 -CH2 3.66/3.60 tiny n.o. no 3.66/3.60-64.90 yes no n.o. n.m. Overlapped with Myo-inositol 8 Creatine + Phosphocreatine - CH3 3.02 very large yes 3.92-56.61 yes yes s 3.92-3.02 (COSY) - CH2 3.92 very large yes 3.02-39.66 yes yes s 9 4-Aminobutyric Ac. (GABA) 3.01-1.89 (COSY) 1 -CH 3.01 medium yes 3.01-42.03 yes yes t 7.58 Overlapped with Carnosine 2 3.01-2.28 (TOCSY) 2 -CH2 1.89 medium yes 2.28-37.14 yes yes q 7.36 Overlapped with (possible) acetic acid 2.28-1.89 (COSY) 3 -CH2 2.28 medium yes 1.89-26.32 yes yes t 7.42 Overlapped with L-Valine (very tiny) 10 Glucose Alpha-D-Glucose 1- CH 5.22 tiny n.o.
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