Supporting Information Proteomic Analyses of Cysteine Redox in High-fat-fed and Fasted Mouse Livers: Implications for Liver Metabolic Homeostasis Yixing Li 1#, Zupeng Luo 1#, Xilong Wu 2, Jun Zhu 2, Kai Yu 1, Yi Jin 1, Zhiwang Zhang 1, Shuhong Zhao 3, Lei Zhou 1* 1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Animal Science and Technology, Guangxi University, Nanning 530004, P.R. China 2 Jingjie PTM Biolab Co. Ltd., Hangzhou Economic and Technological Development Area, Hangzhou 318, P.R. China 3 Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, P. R. China. Table of Contents Table S-1: Group packet information of samples and sub-samples.s-2 Table S-2: IodoTMT (6-plex) labeling of sub-samples.s-3 Table S-3: The GO terms of level 2 distribution (F vs CK).S-4 Table S-4: The subcellular location (F vs CK)..S-6 Figure S-1: Hepatic TG levels in the mouse liver... S-7 Figure S-2: GO-based enrichment analysis (F vs CK) S-8 Figure S-3: KEGG pathway-based enrichment analysis (F vs CK)..S-9 Figure S-4: Domain enrichment analysis (F vs CK)......S-10 Figure S-5: Protein complex enrichment analysis (F vs CK) S-11 Figure S-6: Altered cysteine oxidation of catalytic enzymes in carbon metabolism processes..s-12 S-1
Table S-1. Group Packet Information of Samples and Sub-samples. Groups Sample names Sub-sample Group 1 Group 2 CK1 HF1 F1 CK2 HF2 F2 CK1_Whole CK1_Switch HF1_Whole HF1_Switch F1_Whole F1_Switch CK2_Whole CK2_Switch HF2_Whole HF2_Switch F2_Whole F2_Switch S-2
Table S-2. IodoTMT (6-plex) Labeling of Sub-samples. Group Sub-samples Labels Group 1 CK1_Whole 126 CK1_Switch 127 HF1_Whole 128 HF1_Switch 129 F1_Whole 130 F1_Switch 131 CK2_Whole 126 CK2_Switch 127 Group 2 HF2_Whole 128 HF2_Switch 129 F2_Whole 130 F2_Switch 131 Labels 126-131 are components of iodotmtsixplex Isobaric Label Reagent Set, a product of Thermo Scientific. During the MS/MS stage of acquisition to derive fragment ions and sequence information, a unique reporter ion mass is generated (e.g., 126-131Da for the iodotmt 6 Isobaric Label Reagents). S-3
Table S-3. The GO (Gene Ontology) Terms of Level 2 Distribution of Both Up-regulated (A) and Down-regulated (B) Redox Cysteine Proteins (F vs CK). A. GO Terms of Level 2 Distribution of Up-regulated Redox Cysteine Proteins (F vs CK). GO Terms Level 1 GO Terms Level 2 No. of Protein Biological Process metabolic process 15 cellular process 15 single-organism process 12 developmental process 6 response to stimulus 6 multicellular organismal process 6 biological regulation 6 localization 5 cellular component organization or biogenesis 5 other 4 Cellular Component cell 17 organelle 14 membrane 9 macromolecular complex 3 extracellular region 3 membrane-enclosed lumen 3 extracellular matrix 1 Molecular Function catalytic activity 15 binding 13 transporter activity 4 S-4
B. GO Terms of Level 2 Distribution of Down-regulated Redox Cysteine Proteins (F vs CK). GO Terms Level 1 GO Terms Level 2 No. of Protein Biological Process cellular process 231 metabolic process 224 single-organism process 134 biological regulation 97 response to stimulus 89 localization 64 multicellular organismal process 63 cellular component organization or biogenesis 54 developmental process 46 signaling 25 other 50 Cellular Component cell 257 organelle 230 membrane 116 macromolecular complex 91 membrane-enclosed lumen 61 extracellular region 35 other 20 Molecular Function binding 196 catalytic activity 182 structural molecule activity 17 transporter activity 14 electron carrier activity 11 other 18 S-5
Table S-4. The Subcellular Location of Both Up-regulated and Down-regulated Redox Cysteine Proteins (F vs CK). A. Subcellular Location of Up-regulated Redox Cysteine Proteins (F vs CK). Subcellular Location No. of Protein cyto 10 E.R. 1 extr 4 mito 2 cyto_nucl 1 nucl 2 B. Subcellular Location of Down-regulated Redox Cysteine Proteins (F vs CK). Subcellular Location No. of Protein mito 75 extr 39 cyto 110 E.R. 10 plas 12 nucl 23 pero 2 cyto_nucl 8 cysk 3 mito: Mitochondria; extr: Extracellular; cyto: Cytosol; E.R.: Endoplasmic Reticulum; plas: Plasma Membrane; nucl: Nuclear; pero: Peroxisome; cyto_nucl: Both in Cytosol and Nuclear; cysk: Cytoskeleton. S-6
Relative Liver TG Levels 6 4 2 0 ** ** CK HF F Figure S-1. Hepatic TG levels in the mouse liver (CK, control group; HF, high-fat-feeding group; F, fasting group). **p < 0.01, Student s t test. S-7
A Cellular Component Molecular Function Biological Process cytoplasmic part mitochondrion microbody Golgi apparatus catalytic activity drug binding carboxylic acid binding transferase activity, transferring acyl groups small molecule metabolic process oxidation-reduction process oxoacid metabolic process lipid metabolic process small molecule biosynthetic process organic acid catabolic process cellular lipid metabolic process 0 1 2 3 4 5 6 7 8 9 3.89 3.6 2.49 2.28 2.28 1.54 1.47 1.36 3.9 3.07 2.83 2.49 2.19 2 1.71 1.54 4.8 4.53 4.34 4.3 3.83 3.79 3.7 3.64 3.64 3.63 3.53 3.35 7.74 6.67 B Molecular Function Cellular Component Biological Process oxidoreductase activity, acting on CH-OH oxidoreductase activity, acting on the CH- catalytic activity small molecule binding mitochondrial inner membrane cytosol mitochondrial envelope cytoplasmic part dicarboxylic acid metabolic process organic acid catabolic process organic substance biosynthetic process monocarboxylic acid catabolic process single-organism metabolic process cellular metabolic process primary metabolic process 0 20 40 60 80 120 27.16 12.54 12.35 11.65 60.94 57.42 54.67 42.58 33.96 29.99 26.68 14.02 Figure S-2. GO-based enrichment analysis of up-regulated (A) and down-regulated (B) proteins (F vs CK). The numerical bars represent the results of -log10(fisher s exact test p value). The higher the numbers, the lower the p values and the more significant the differentially regulated redox cysteine proteins are enriched to this function. S-8
A mmu01 Metabolic pathways - Mus musculus (mouse) mmu01230 Biosynthesis of amino acids - Mus musculus (mouse) 0 0.5 1 1.5 2 2.5 3 3.5 4 2.16 3.6 B mmu01 Metabolic pathways - Mus musculus mmu00280 Valine, leucine and isoleucine mmu01200 Carbon metabolism - Mus musculus mmu00071 Fatty acid degradation - Mus mmu01230 Biosynthesis of amino acids - Mus mmu00020 Citrate cycle (TCA cycle) - Mus mmu00620 Pyruvate metabolism - Mus musculus mmu00350 Tyrosine metabolism - Mus musculus mmu01212 Fatty acid metabolism - Mus mmu00640 Propanoate metabolism - Mus mmu00650 Butanoate metabolism - Mus mmu00630 Glyoxylate and dicarboxylate mmu05012 Parkinson's disease - Mus musculus mmu00380 Tryptophan metabolism - Mus mmu00250 Alanine, aspartate and glutamate mmu00330 Arginine and proline metabolism - mmu00270 Cysteine and methionine metabolism - mmu00010 Glycolysis / Gluconeogenesis - Mus mmu04146 Peroxisome - Mus musculus (mouse) mmu00410 beta-alanine metabolism - Mus mmu00260 Glycine, serine and threonine mmu03320 PPAR signaling pathway - Mus mmu03010 Ribosome - Mus musculus (mouse) mmu00190 Oxidative phosphorylation - Mus mmu05016 Huntington's disease - Mus musculus Figure S-3. KEGG pathway-based enrichment analysis of up-regulated (A) and down-regulated (B) proteins (F vs CK). The numerical bars represent the results of -log10(fisher s exact test p value). The higher the numbers, the lower the p values and the more significant the differentially regulated redox cysteine proteins are enriched to this pathway. 0 5 10 15 20 25 30 35 40 13.9 13.58 13.11 12.02 10.96 8.73 8.68 8.52 8.34 6.91 5.67 5.54 5.48 5.4 5.37 5.23 5.09 5.07 4.86 4.25 4.13 4.08 4.01 3.76 37.85 S-9
A 0 0.5 1 1.5 2 2.5 3 Serum albumin, N-terminal Serum albumin-like Thiolase-like Thiolase-like, subgroup Aldolase-type TIM barrel 1.31 1.86 1.79 2.37 2.52 B NAD(P)-binding domain Acyl-CoA dehydrogenase/oxidase, N-terminal Acyl-CoA dehydrogenase/oxidase C-terminal Acyl-CoA oxidase/dehydrogenase, central domain Acyl-CoA dehydrogenase/oxidase, N-terminal Aldehyde dehydrogenase, C-terminal Aldehyde dehydrogenase domain Aldehyde dehydrogenase, N-terminal Aldehyde/histidinol dehydrogenase Thiolase-like Thiolase-like, subgroup Pyridoxal phosphate-dependent transferase, Thiolase, C-terminal Pyridoxal phosphate-dependent transferase Pyridoxal phosphate-dependent transferase, Dehydrogenase, multihelical Thiolase, N-terminal 6-phosphogluconate dehydrogenase, C- Alcohol dehydrogenase, C-terminal GroEL-like apical domain Pre-ATP-grasp domain Acyl carrier protein-like ATP-grasp fold, subdomain 2 GroEL-like equatorial domain GroES (chaperonin 10)-like 0 2 4 6 8 10 12 14 7.39 7.16 6.96 6.86 6.86 6.42 6.42 6.27 5.91 5.54 4.21 4.03 3.9 3.84 3.69 3.43 3.28 3.15 3.03 2.73 2.71 2.65 2.65 2.57 11.57 Figure S-4. Domain enrichment analysis of up-regulated (A) and down-regulated (B) proteins (F vs CK). The numerical bars represent the results of -log10(fisher s exact test p value). The higher the numbers, the lower the p values and the more significant the differentially regulated redox cysteine proteins are enriched to this domain. S-10
0 1 2 3 4 5 6 Parvulin-associated pre-rrnp complex Respiratory chain complex I, mitochondrial Profilin 1 complex CCT complex CCT micro-complex CCT complex, testis specific 1.72 1.67 1.42 1.42 1.42 4.97 Figure S-5. Protein complex enrichment analysis of down-regulated proteins (F vs CK). The numerical bars represent the results of -log10(fisher s exact test p value). The higher the numbers, the lower the p values and the more significant the differentially regulated redox cysteine proteins are enriched to this complex. S-11
Figure S-6. Altered cysteine oxidation of catalytic enzymes in central carbon metabolism processes upon fasting. A variety of enzymes with regulated redox cysteine sites were marked in the major metabolic pathways such as TCA cycle, glycolysis/gluconeogenesis, oxidative phosphorylation and fatty acid oxidation. The blue and red color represents down- and up-regulated cysteine oxidation of each enzyme, respectively. S-12