TABLE 1.
List of potential Cgi-58 interaction partners identified by label transfer experiments with subsequent nano-LC/MS-MS analysis
Purified Gst-tagged Cgi-58 was labeled with the heterobifunctional cross-linker Sulfo-SBED and incubated with murine WAT lysates to label interacting proteins with a biotin tag. After affinity purification, the interacting proteins were separated by SDS-PAGE, tryptically digested in gel, and analyzed with nano-LC-MS/MS. Proteins found in the respective samples including the NCBI accession numbers, their molecular masses, the number of identified distinct peptides, the Spectrum Mill protein score, and the percentage of amino acid coverage are given in the table. The database used for re-analysis of the raw data was the “mouse” subset of the “SwissProt” database, downloaded on 7.1.2014 from “ncbi.nih.gov” (456,996 total entries). Database searching was performed with the “MS/MS Search” feature of the “Agilent-Spectrum Mill” software (revision 2.7). Acceptance parameter: individual peptide score: min 10; protein score: min 20, and minimum number of distinct peptides: 2. Keratin was excluded from the list.
| Spectra | Distinct peptides | Distinct summed MS/MS search score | % amino acid coverage | Protein mass | Species | Database accession no. | Protein name |
|---|---|---|---|---|---|---|---|
| Da | |||||||
| Sample 1 | |||||||
| 33 | 6 | 90.5 | 20 | 29,366.4 | Mouse | P16015.3 | Carbonic anhydrase 3 |
| 6 | 5 | 70.35 | 22 | 24,870.8 | Mouse | O08709.3 | Peroxiredoxin-6 |
| 6 | 4 | 57.09 | 14 | 25,970.2 | Mouse | P10649.2 | Glutathione S-transferase Mu 1 |
| 6 | 4 | 51.08 | 5 | 68,693 | Mouse | P07724.3 | Serum albumin; Flags, precursor |
| 3 | 3 | 46.96 | 11 | 27,623.4 | Mouse | Q9DCW4.3 | Electron transfer flavoprotein subunit β; short = β-ETF |
| 6 | 3 | 46.64 | 10 | 28,086.6 | Mouse | Q9CQV8.3 | 14-3-3 protein β/α |
| 3 | 3 | 45.14 | 12 | 23,013.9 | Mouse | P14602.3 | Heat shock protein β-1; short = HspB1 |
| 4 | 3 | 43.9 | 12 | 28,832.1 | Mouse | Q9DBJ1.3 | Phosphoglycerate mutase 1 |
| 3 | 2 | 29.75 | 6 | 32,191.9 | Mouse | P17751.4 | Triosephosphate isomerase; short = TIM |
| 3 | 2 | 29.15 | 18 | 14,650 | Mouse | P04117.3 | Fatty acid-binding protein, adipocyte |
| 4 | 2 | 29.09 | 7 | 30,615.7 | Mouse | Q00623.2 | Apolipoprotein A-I; short = Apo-AI; short = ApoA-I |
| 3 | 2 | 26.78 | 7 | 26,468.9 | Mouse | Q9WTP6.5 | Adenylate kinase 2, mitochondrial; short = AK 2 |
| 4 | 2 | 26.28 | 7 | 27,679.8 | Mouse | Q03401.1 | Cysteine-rich secretory protein 1; short = CRISP-1 |
| 4 | 2 | 24.81 | 4 | 53,951.8 | Mouse | Q2KHK6.2 | Gasdermin-C2 |
| 3 | 2 | 23.57 | 1 | 217,752.2 | Mouse | Q6PDQ2.1 | Chromodomain-helicase-DNA-binding protein 4; short = CHD-4 |
| 3 | 2 | 23.4 | 3 | 33,267.1 | Mouse | Q8R2Z0.2 | Protein FAM132A; Flags: precursor |
| 4 | 2 | 22.66 | 2 | 117,772.7 | Mouse | Q9JHR7.1 | Insulin-degrading enzyme |
| 3 | 2 | 20.89 | 0 | 833,642.4 | Mouse | Q91ZU6.1 | Dystonin |
| 3 | 2 | 20.85 | 8 | 37,549 | Mouse | O35864.3 | COP9 signalosome complex subunit 5; short = SGN5; short = signalosome subunit 5 |
| 3 | 2 | 20.64 | 0 | 292,121 | Mouse | Q8C170.2 | Unconventional myosin-IXa |
| 2 | 2 | 20.22 | 0 | 390,6506.8 | Mouse | A2ASS6.1 | Titin |
| 2 | 2 | 20.09 | 0 | 282,962.5 | Mouse | Q5DU37.2 | Zinc finger FYVE domain-containing protein 26 |
| Sample 2 | |||||||
| 16 | 8 | 117.71 | 51 | 15,878.3 | Mouse | P02089.2 | Hemoglobin subunit β-2 |
| 20 | 5 | 87.34 | 34 | 14,650 | Mouse | P04117.3 | Fatty acid-binding protein, adipocyte |
| 26 | 5 | 78.53 | 31 | 15,085.2 | Mouse | P01942.2 | Hemoglobin subunit α |
| 8 | 3 | 53.7 | 15 | 22,176.6 | Mouse | P35700.1 | Peroxiredoxin-1 |
| 4 | 3 | 50.67 | 11 | 29,366.4 | Mouse | P16015.3 | Carbonic anhydrase 3 |
| 4 | 3 | 47.96 | 24 | 13,992.3 | Mouse | Q8CGP2.3 | Histone H2B type 1-P |
| 4 | 4 | 46.9 | 0 | 3,906,506.8 | Mouse | A2ASS6.1 | Titin |
| 3 | 3 | 46.02 | 18 | 20,830.5 | Mouse | P70296.3 | Phosphatidylethanolamine-binding protein 1; short = PEBP-1 |
| 9 | 3 | 45.27 | 26 | 14,161.6 | Mouse | Q8CGP5.3 | Histone H2A type 1-F |
| 5 | 3 | 44.97 | 17 | 17,971.4 | Mouse | P17742.2 | Peptidyl-prolyl cis-trans isomerase A; short = PPIase A |
| 3 | 2 | 37.14 | 13 | 21,897.6 | Mouse | P99029.2 | Peroxiredoxin-5, mitochondrial |
| 4 | 2 | 29.5 | 13 | 15,137.5 | Mouse | Q05816.3 | Fatty acid-binding protein, epidermal |
| 3 | 3 | 28.28 | 22 | 36,347.9 | Mouse | Q8C0D0.1 | Probable tRNA pseudouridine synthase 1 |
| 9 | 2 | 27.38 | 3 | 79,574.8 | Mouse | Q8R322.2 | Nucleoporin GLE1 |
| 3 | 3 | 24.98 | 0 | 55,0837 | Mouse | Q9QYX7.4 | Protein piccolo |
| 3 | 2 | 24.85 | 14 | 22,395.6 | Mouse | Q9WVA4.4 | Transgelin-2 |
| 3 | 2 | 23.5 | 1 | 245,759.3 | Mouse | Q8BZN6.3 | Dedicator of cytokinesis protein 10 |
| 3 | 2 | 22.18 | 1 | 281,223 | Mouse | Q8BTM8.5 | Filamin-A; short = FLN-A |
| 2 | 2 | 22.02 | 0 | 519,211.7 | Mouse | A2ARV4.1 | Low-density lipoprotein receptor-related protein 2; short = LRP-2 |
| 3 | 2 | 21.65 | 6 | 40,489.2 | Mouse | P08752.5 | Guanine nucleotide-binding protein Gi subunit α-2 |
| 4 | 2 | 21.08 | 1 | 157,204.6 | Mouse | O09053.3 | Werner syndrome ATP-dependent helicase homolog |
| 2 | 2 | 20.17 | 2 | 103,938 | Mouse | Q6P9P8.1 | DENN domain-containing protein 2C |
| Sample 3 | |||||||
| 10 | 6 | 101.96 | 55 | 15,840.3 | Mouse | P02088.2 | Hemoglobin subunit β-1 |
| 11 | 5 | 74.76 | 31 | 15,085.2 | Mouse | P01942.2 | Hemoglobin subunit α |
| 6 | 5 | 57.86 | 28 | 17,971.4 | Mouse | P17742.2 | Peptidyl-prolyl cis-trans isomerase A; short = PPIase A |
| 3 | 2 | 35.84 | 16 | 14,650 | Mouse | P04117.3 | Fatty acid-binding protein, adipocyte |
| 3 | 2 | 35.5 | 14 | 22,395.6 | Mouse | Q9WVA4.4 | Transgelin-2 |
| 4 | 2 | 33.14 | 19 | 13,994.3 | Mouse | Q9D2U9.3 | Histone H2B type 3-A |
| 4 | 2 | 32.25 | 21 | 14,161.6 | Mouse | Q8CGP5.3 | Histone H2A type 1-F |
| 2 | 2 | 31.95 | 15 | 18,559.7 | Mouse | P18760.3 | Cofilin-1 |
| 2 | 2 | 31.74 | 11 | 20,830.5 | Mouse | P70296.3 | Phosphatidylethanolamine-binding protein 1; short = PEBP-1 |
| 3 | 2 | 31.41 | 13 | 15,137.5 | Mouse | Q05816.3 | Fatty acid-binding protein, epidermal |
| 3 | 3 | 30.13 | 16 | 61,422.6 | Mouse | Q61207.2 | Sulfated glycoprotein 1; short = SGP-1 |
| 5 | 2 | 29.13 | 11 | 14,865.9 | Mouse | P16045.3 | Galectin-1; short = Gal-1 |
| 3 | 3 | 28.14 | 0 | 3,906,506.8 | Mouse | A2ASS6.1 | Titin |
| 4 | 2 | 25.17 | 13 | 15,942.8 | Mouse | P08228.2 | Superoxide dismutase (CuZn) |
| 4 | 2 | 24.57 | 17 | 10,962.8 | Mouse | Q64433.2 | 10 kDa heat shock protein, mitochondrial; short = Hsp10 |
| 7 | 2 | 23.08 | 3 | 167,141.5 | Mouse | Q925J9.2 | Mediator of RNA polymerase II transcription subunit 1 |
| 5 | 2 | 23 | 3 | 79,574.8 | Mouse | Q8R322.2 | Nucleoporin GLE1 |
| 2 | 2 | 22.91 | 11 | 32,970.4 | Mouse | O35075.1 | Down syndrome critical region protein 3 homolog |
| 2 | 2 | 22.74 | 4 | 62,700.3 | Mouse | Q9JHU2.1 | Palmdelphin |
| 2 | 2 | 22.06 | 6 | 40,489.2 | Mouse | P08752.5 | Guanine nucleotide-binding protein Gi subunit α-2 |
| 3 | 2 | 21.9 | 1 | 244,562.5 | Mouse | A2AGH6.1 | Mediator of RNA polymerase II transcription subunit 12 |
| 3 | 2 | 21.29 | 2 | 124,935 | Mouse | Q6A025.2 | Protein phosphatase 1 regulatory subunit 26 |
| 10 | 2 | 20.4 | 1 | 157,204.6 | Mouse | O09053.3 | Werner syndrome ATP-dependent helicase homolog |
| 2 | 2 | 20.07 | 3 | 96294.8 | Mouse | Q8C9H6.1 | Striatin-interacting proteins 2 |
| 2 | 2 | 20 | 0 | 392,324.8 | Mouse | Q9JJN2.1 | Zinc finger homeobox protein 4 |
| Sample 4 | |||||||
| 61 | 11 | 205.71 | 88 | 15,840.3 | Mouse | P02088.2 | Hemoglobin subunit β-1 |
| 32 | 9 | 170.54 | 60 | 14,650 | Mouse | P04117.3 | Fatty acid-binding protein, adipocyte |
| 14 | 7 | 117.46 | 30 | 17,971.4 | Mouse | P17742.2 | Peptidyl-prolyl cis-trans isomerase A; short = PPIase A |
| 8 | 6 | 114.55 | 29 | 29,366.4 | Mouse | P16015.3 | Carbonic anhydrase 3 |
| 44 | 6 | 103.08 | 42 | 15,085.2 | Mouse | P01942.2 | Hemoglobin subunit α |
| 8 | 4 | 73.3 | 28 | 21,897.6 | Mouse | P99029.2 | Peroxiredoxin-5, mitochondrial |
| 4 | 4 | 61.97 | 47 | 13,159.6 | Mouse | Q9Z0F7.1 | γ-Synuclein |
| 6 | 3 | 60 | 25 | 15,942.8 | Mouse | P08228.2 | Superoxide dismutase (CuZn) |
| 7 | 4 | 59.11 | 30 | 14,865.9 | Mouse | P16045.3 | Galectin-1; short = Gal-1 |
| 8 | 3 | 56.68 | 28 | 20,830.5 | Mouse | P70296.3 | Phosphatidylethanolamine-binding protein 1; short = PEBP-1 |
| 4 | 3 | 50.09 | 25 | 15,776 | Mouse | P07309.1 | Transthyretin |
| 5 | 3 | 50 | 4 | 68,693 | Mouse | P07724.3 | Serum albumin; Flags: precursor |
| 6 | 3 | 48.45 | 14 | 15,137.5 | Mouse | Q05816.3 | Fatty acid-binding protein, epidermal |
| 4 | 4 | 39.37 | 0 | 3,906,506.8 | Mouse | A2ASS6.1 | Titin |
| 3 | 2 | 36.31 | 29 | 11,082.8 | Mouse | P50543.1 | Protein S100-A11 |
| 3 | 2 | 36.14 | 3 | 82,550.5 | Mouse | P0CG50.2 | Polyubiquitin-C; contains: ubiquitin; contains: ubiquitin-related 1; contains: ubiquitin-related 2; Flags: precursor |
| 3 | 2 | 33.74 | 21 | 18,559.7 | Mouse | P18760.3 | Cofilin-1 |
| 3 | 2 | 31.27 | 19 | 14,255.5 | Mouse | P52760.3 | Ribonuclease UK114 |
| 4 | 3 | 26.9 | 6 | 113,100.4 | Mouse | P11103.3 | Poly [ADP-ribose] polymerase 1; short = PARP-1 |
| 2 | 2 | 25.62 | 23 | 10,962.8 | Mouse | Q64433.2 | 10 kDa heat shock protein, mitochondrial; short = Hsp10 |