Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS_DMSO_BR3_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS_MG132_BR1_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS_DMSO_BR1_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_MG132_BR2_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS12221099_DMSO_BR2_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_DMSO_BR3_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_MG132_BR3_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_DMSO_BR4_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS12221099_DMSO_BR4_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_MG132_BR4_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_DMSO_BR1_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS_MG132_BR4_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS12221099_MG132_BR3_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_DMSO_BR4_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS_DMSO_BR1_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS_MG132_BR2_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS_MG132_BR4_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS12221099_MG132_BR4_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS12221099_DMSO_BR3_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_MG132_BR4_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_MG132_BR4_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_DMSO_BR1_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS12221099_MG132_BR2_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_DMSO_BR2_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_MG132_BR2_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS12221099_DMSO_BR3_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_MG132_BR4_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_MG132_BR2_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS12221099_MG132_BR5_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_MG132_BR1_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_MG132_BR3_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_DMSO_BR2_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS_MG132_BR3_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_DMSO_BR1_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS12221099_DMSO_BR1_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_DMSO_BR3_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_DMSO_BR3_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS12221099_MG132_BR1_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS_DMSO_BR4_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS_MG132_BR1_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_DMSO_BR2_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_MG132_BR3_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS_MG132_BR3_TR1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS12221099_DMSO_BR4_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_US_U2OS12221099_MG132_BR3_TR3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_MG132_BR1_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_double_SU_U2OS_MG132_BR5_TR2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/09/PXD007733/QE_SC_d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CuijpersAffinity purification coupled with mass spectrometry proteomicsMass SpectrometryShotgun proteomicsNot availableHumanUbiquitinSumoNanolc-ms/msCo-modificationPtmshttp://www.ebi.ac.uk/pride/archive/projects/PXD007733For each sample 200 million cells were lysed in 20 ml His10-pulldown lysis buffer (6 M guanidine-HCl, 100 mM Na2HPO4/NaH2PO4 (pH 8.0) and 10 mM Tris-HCl (pH 8.0)), stored at -80°C and thawed at RT to continue sample preparation. After sonicating twice for 10 seconds at 30 Watts, samples were equalized using BCA Protein Assay Reagent (Thermo Scientific). Imidazole (pH 8.0, Merck Millipore) and β-mercaptoethanol (Sigma) were added to a final concentration of 50 mM and 5.0 mM, respectively. Samples were mixed for 15 minutes at room temperature, before adding 20 µl of Ni-NTA beads per 1 ml of lysis buffer and incubation overnight at 4°C under continuous rotation. Subsequently, the beads were washed once with buffer 1, once with buffer 2, once with buffer 3 and twice with buffer 4 including three tube changes. Wash buffer 1 consisted of His10-pulldown lysis buffer supplemented with 10 mM imidazole (pH 8.0), 5 mM β-mercaptoethanol and 0.1% Triton X-100. Wash buffer 2 contained 8 M urea, 100 mM Na2HPO4/NaH2PO4 (pH 8.0), 10 mM Tris-HCl (pH 8.0), 10 mM imidazole (pH 8.0), 5 mM β-mercaptoethanol and 0.1% Triton X-100. Wash buffer 3 consisted of 8 M urea, 100 mM Na2HPO4/NaH2PO4 (pH 6.3), 10 mM Tris-HCl (pH 6.3), 10 mM imidazole (pH 7.0) and 5 mM β-mercaptoethanol. Wash buffer 4 contained 8 M urea, 100 mM Na2HPO4/NaH2PO4 (pH 6.3), 10 mM Tris-HCl (pH 6.3) and 5 mM β-mercaptoethanol. Samples were eluted three times at RT with 400 µl elution buffer (7 M urea, 100 mM Na2HPO4/NaH2PO4, 10 mM Tris-HCl (pH 7.0), 500 mM imidazole (pH 7.0) and 70 mM chloroacetamide (CAA)). The combined elution was passed through pre-washed 0.45 µm filter columns (Millipore) to remove any residual beads and subsequently concentrated on pre-washed 100 kDa cut off filters (SartoriusStedim). Sample volumes were equalized to 25.0 µl, before taking 10% of the samples as pulldown controls for immunoblot analysis. The remaining part of the sample was slowly and step-wise diluted on ice in 50 mM Na2HPO4/NaH2PO4 (pH 7.0), 10 mM Tris-HCl (pH 7.0), 100 mM NaCl and 70 mM CAA to a final volume of 1 ml. After centrifugation for 45 minutes at 13200 rpm and 4°C, the supernatant was transferred to a new tube and 100 µl of anti-FLAG-M2 beads (Sigma) was added per sample. NuPAGE LDS Sample Buffer (LDS, Life technologies) was added to solubilize any potential proteins in the pellet as a control for the renaturation step. After incubation of the supernatant with the beads for 90 minutes at 4°C, samples were washed twice with buffer 5, twice with buffer 6 and three times with buffer 7 including three tube changes. Buffer 5 consisted of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl and 70 mM CAA. Buffer 6 contained 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl, while buffer 7 consisted of 50 mM ammonium bicarbonate (ABC, Sigma). As a control for immunoblot analysis, 10% of the beads was eluted in LDS. The remaining beads were resuspended in 100 µl buffer 7 supplemented with 0.25 µg trypsin (Promega) per sample and incubated overnight at 37°C. Samples were passed through pre-washed 0.45 µm filter columns to remove the beads, before trifluoroacetic acid (TFA, Sigma) was added to a final concentration of 2% to acidify the samples. Stage tips containing C18 (Sigma) were activated by passing HPLC-grade methanol (Sigma), washed with 80% acetonitrile (ACN, Sigma) in 0.1% formic acid (FA, Sigma) and equilibrated with 0.1% FA. Subsequently, the samples were loaded on these stage tips. Upon washing twice with 0.1% FA, the stage tips were dried completely and eluted twice with 80% ACN. The samples were vacuum dried using a SpeedVac RC10.10 (Jouan), redissolved in 0.1% FA and transferred to autoloader vials before measurement by mass spectrometry.PrideNot availablemonohydroxylated residueacetylated residueFor each experimental condition at least four biological replicates were performed to allow detection of significant differences, which were all measured in technical triplicate by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Samples were measured on an EASY-nLC 1000 system (Proxeon) connected to an Orbitrap Q-Exactive (Thermo Fisher Scientific) through a nano-electrospray ion source. Peptides were separated in a 13 cm analytical column with an inner-diameter of 75 µm, which was packed in-house with 1.8 µm C18 beads (Reprospher). A gradient length was used of 60 minutes from 2% to 95% ACN in 0.1% FA with a flow rate of 200 nl/minute. The data-dependent acquisition mode with a top 10 method was used to operate the mass spectrometer. Full-scan MS spectra were acquired at a target value of 3 x 106 with a resolution of 70000. The higher-collisional dissociation tandem mass spectra were recorded at a target value of 1 x 105 and a resolution of 17500 with a normalized collision energy of 25%. The maximum injection times for MS1 and MS2 were respectively 20 ms and 100 ms. For 60 seconds, the precursor ion masses of scanned ions were dynamically excluded from MS/MS analysis. Ions with a charge of 1 or greater than 6 were excluded from triggering MS2 events. Subsequently, the raw data analysis was performed using MaxQuant Software version 1.5.3.30 with its integrated search engine Andromeda. A first search was carried out with 20 ppm for precursor ions, followed by a main search using 4.5 ppm. To search against the in silico digested proteome containing 92180 entries of Homo sapiens from UniProt (24 March 2016), the mass tolerance of MS/MS spectra were set to 20 ppm. In addition, MS/MS data were searched by Andromeda for potential common mass spectrometry contaminants. Trypsin/P specificity was used to perform database searches, allowing four missed cleavages. In addition, carbamidomethylation of cysteine residues was considered as a fixed modification, while oxidation of methionines, N-terminal carbamylation and acetylation, and diGly modification on lysines were considered as variable modifications. Match between runs was performed with a 20 minutes alignment time window and a 0.7 minute match time window, while a minimum peptide length of 7 was used. To consider proteins for quantification, at least two identified peptides were required, including unique and razor peptides. Proteins and peptides were identified using a false discovery rate of 1%. Finally, label-free quantification was performed using LFQ settings with fast LFQ disabled to quantify all identified peptides. Since substantial differences between conditions were expected, LFQ normalization by MaxQuant was skipped to prevent undesirable correction between these samples. Proteins identified by the same set of peptides were combined to a single protein group by MaxQuant. The proteins identified in each sample were further analyzed using Perseus Software version 1.5.2.4. Samples from DMSO and MG132 treated cells were analyzed separately to prevent incorrect imputation. Both data sets were filtered for potentially improper protein identifications by removing proteins that would fit the categories ‘potential contaminant’, ‘reverse’ or ‘only identified by site’. Subsequently, all LFQ intensities were log2 transformed and all experimental replicates for each condition were assigned together in four groups per treatment for the main analysis. Finally, all proteins were removed that were not identified in at least four experimental replicates in at least one of these four groups. For an additional tailored analysis in the supplementary data, the experimental conditions of both approaches were pooled together and assigned in two groups per treatment to increase the statistical power. Proteins that were not identified in at least eight pooled replicates of these two groups were removed. For both analyses missing values were imputed based on the total matrix of each data set, using normally distributed values with a randomized 0.3 width (log2) and a 1.8 down shift (log2). Although the samples were not specifically enriched for modified peptides, a search was performed by MaxQuant to identify peptides modified by a diGly motif. Subsequently, all peptides modified by a diGly motif that were identified equally or more in the parental control samples, compared to the samples from cell lines expressing both SUMO2 and ubiquitin, were considered as ubiquitylated background binders and therefore removed from the list. In addition, all peptides assigned to the ubiquitin precursor UBA52 instead of to ubiquitin, or with lower quality spectra were removed to obtain a list containing peptides modified by a diGly motif that were specifically identified in the samples containing co-modified proteins. For each peptide the best localization evidence spectrum was retrieved from MaxQuant.ProteomicsAlfred C.O. VertegaalQ ExactivePARTIALDepartment of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the NetherlandsHomo Sapiens (human)28951443 Cuijpers SAG, Willemstein E, Vertegaal ACO. Converging SUMO and ubiquitin signaling: improved methodology identifies co-modified target proteins. Mol Cell Proteomics. 2017 10.1074/mcp.tir117.000152s.a.g.cuijpers@lumc.nlTechnicalBiologicalLeiden University Medical CenterPost-translational protein modifications (PTMs) including small chemical groups and small proteins, belonging to the ubiquitin family, are essential for virtually all cellular processes. In addition to modification by a single PTM, proteins can be modified by a combination of different modifiers, which are able to influence each other. Because little is known about crosstalk among different ubiquitin family members, we developed an improved method enabling identification of co-modified proteins on a system-wide level using mass spectrometry. We focused on the role of crosstalk between SUMO and ubiquitin during proteasomal degradation. Using two complementary approaches, we identified 498 proteins to be significantly co-modified by SUMO and ubiquitin upon MG132 treatment. These targets included many enzymatic components of PTM machinery, involved in SUMOylation and ubiquitylation, but also phosphorylation, methylation and acetylation, revealing a highly complex interconnected network of crosstalk among different PTMs. In addition, various other biological processes were found to be significantly enriched within the group of co-modified proteins, including transcription, DNA repair and the cell cycle. Interestingly, the latter group mostly consisted of proteins involved in mitosis, including a subset of chromosome segregation regulators. We hypothesize that group modification by SUMO-targeted ubiquitin ligases regulates the stability of the identified subset of mitotic proteins, which ensures proper chromosome segregation. The mitotic regulators KIF23 and MIS18BP1 were verified to be co-modified by SUMO and ubiquitin on inhibition of the proteasome and subsequently identified as novel RNF4 targets. Both modifications on MIS18BP1 were observed to increase simultaneously during late mitosis, whereas the total protein level decreased immediately afterward. These results confirm the regulation of MIS18BP1 via SUMO-ubiquitin crosstalk during mitosis. Combined, our work highlights extensive crosstalk between SUMO and ubiquitin, providing a resource for further unraveling of SUMO-ubiquitin crosstalk.Converging Small Ubiquitin-like Modifier (SUMO) and Ubiquitin Signaling: Improved Methodology Identifies Co-modified Target Proteins.Cuijpers Sabine A G SAG, Willemstein Edwin E, Vertegaal Alfred C O ACOsodium salt, CPD photolyase activity, ammonium formate, 13C-labeled, IL1BC, MeCN, cadmium salt, PLXN4, PhrB photolyase activity, PLXN3, ectodin, CASP-1, Basodexan, magnesium formate, zinc salt, Bca, Trifluoroacetate, hydrogen chloride, cobalt(II) formate dihydrate, GRP1, CH3-C#N, Grp1, B1, imidazole monohydrochloride, HCL-C, Urea, AGM4, 3, NUP96, Analysis, outer pigmented layer of retina, Il1bc, epithelium, bca, ABC, C79325, Analyses, DmelCG13176, PTPSTEP, ORF19, proteins, IL-1BC, HPLC, SUPPRESSOR OF AUXIN RESISTANCE 3, salt, Wasserstoffchlorid, pigment epithelium of retina, anatomical tube, ACN, sample, D1, Acn, Rotations, pigmented retina epithelium, CT, stage, autolysin activity, 6.3, GPH, nickel salt, 0610006G05Rik, renaturation, Caspase-1 subunit p10, cobalt (+2) salt, familial, Ct, Guanidine Monohydroiodine, Spectrum Analysis, DmelCG7788, common salt, sodium (4:1:1) salt, magnesium salt, Chlorwasserstoff, dipyrimidine photolyase (photosensitive), 1110049F14Rik, leukemic reticuloendotheliosis, Clinorotations, Acid, CG11628, LY57, formate, Spectrometry, Guanidine Phosphate, reagent, cromium (+3), Striatum-enriched protein-tyrosine phosphatase, beta Trypsin, spirit of wood, duct, Wise, Drice, bacteriolytic toxin activity, ammonium (4:1) salt, methyl cyanide, chlorane, DRICE, lead (+2) salt, Methyl Alcohol, umat, 2-ME, lifespan, l(1)VE614, GRP1/cytohesin 1, nickel formate dihydrate, lead formate, chlorure d'hydrogene, DrIce, DrICE, developmental stage, aluminum salt, Carbamide, CH3OH, DUH3, deoxyribodipyrimidine photolyase activity, stratum pigmentosum retinae, CG11633, Guanidium, Ly57, Gene Products, H2im(+), Carmol, Cesium Trifluoroacetate, Monohydroiodine, FAM39E, 2310061K05Rik, Acinus, Hydrogen chloride, thallium (+1) salt, PCE-2, beta-Trypsin, rubidium salt, F23A5.3, methanoic acid, kf, acinusL, Monohydrobromide, Guanidinium Chloride, hairy cell leukemia, Caspase-1 subunit p20, acinusS, Kochsalz, LAN, Al-imidazole, PTHB1, Guanidine Monohydrate, Guanidine Nitrate, 1-(14)C-labeled, caspase 3, Proteins, stepk, Methyl alcohol, chloridohydrogen, potassium formate, CE-2, copper (+2) salt, acetonitrile, buffer, Cell, mKIAA0670, Wash2, Wash1, Temperatures, Natriumchlorid, carbonyldiamide, C18, cupric formate, chemical analysis, Nitrate, Sodium, MeOH, NaCl, aluminum formate, dJ142L7.2, Mass Spectrum Analyses, Guanidine Monohydrobromide, Guanidine, RPE, lithium formate, holin, Guanidine Sulfate, Chloride, IL-1 beta-converting enzyme, ethanenitrile, imidazolium, Interleukin-1 beta-converting enzyme, retinal pigment epithelium, DmelCG11628, p. pigmentosa retinae, Gene Proteins, 2-mercapto-, deoxyribonucleate pyrimidine dimer lyase (photosensitive), Sulfate, hydrochloric acid, Methoxide, imidazole acetate, NCMe, determination, classic hairy cell leukaemia, zinc formate, CG7788, Monohydrate, crice, Guanidine Sulfate (2:1), lead salt, Hydrogenchlorid, lysis, pigmented epithelium, HCHWA, Cut, Ethanol, halite, ammonium tetraformate, uninterrupted, BANF, l(1)7Ba, l(1)7Bb, wood alcohol, Mass Spectrum Analysis, kDa, reference sample, entire life cycle, E927b, 2610036I19Rik, 2610510L13Rik, pigmented retina, 2 Mercaptoethanol, USAG-1, Phosphate, ABC14, copper, DNA cyclobutane dipyrimidine photolyase activity, CG11387, genetic, cloruro sodico, fSAP152, lithium salt, ICE, l(2)k08110, HSSEXGENE, trifluoro-, MCOPCB7, necrosis, Guanidine Sulfate (1:1), Lyw-57, Sodium Methoxide, MTABC3, ammonium (2:1) salt, reagents, PRE, Tube, rock salt, XAP-6, Monohydrochloride, Alcohol, ice, iCE, drice, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, PRP, classic hairy cell leukemia, pigmented retinal epithelium, Thermo Scientific, ur, hereditary cerebral hemorrhage with amyloidosis - Dutch type, Spectroscopy, SLP65, TUBE, Ly-57, carbamide, imidazole conjugate monoacid, retinal pigment, drIce, drICE, 3.1.3.48, HCL, bacteriocin activity, WASH, BcDNA:GH10590, reactif, nickel (+2) salt, Carbinol, retinal pigment layer, Acetic acid, carbinol, SLP-65, ammonium salt, BASH, Step, retinal pigmented epithelium, life, HCl, P45, Trifluoroacetic, cobaltous formate, Guanidine Monohydrochloride, natrii chloridum, chloracetamide, Clinorotation, METHANOL, H2NC(O)NH2, phr A photolyase activity, Wood, lysin activity, imidazolium chloride, rotation, DNA-photoreactivating enzyme, Cesium, STEP, Sostl, 2-Mercaptoethanol, copper salt, p45, imidazolium ion, 1728, IMIDAZOLE, dutch hereditary cerebral amyloid angiopathy, cesium salt, Gene, Methyl, ACINUS, Spectrum Analyses, photoreactivating enzyme activity, Methylalkohol, Buffer, formic acid, Karbamid, cytohesin/GRP1, potassium salt, p63, 14C-labeled, p65, Harnstoff, Mass, imidazole citrate, Wood Alcohol, stratum pigmentosa retinae, cloruro de hidrogeno, Mass Spectroscopy, Pro-Mega, Guanidium Chloride, MOS3, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, reactivo, PRECOCIOUS, entire lifespan, l(2)SH2 0323, chlorure de sodium, methanol, calcium formate, Neural-specific protein-tyrosine phosphatase, DmelCG11387, wood naphtha, Cu-imidazole, cromium (+3) salt, imidazole sodium, Guanidine Hydrochloride, F23A5_3, sodium formate, BLNK-S, imidazole monophosphonate, Controlled, Controlling, nickel formate, 3H-labeled, TSH1, strontium formate, NY-CO-33, strontium salt, Guanidinium, Methanol, CAA, [HCl], stratum pigmentosum (retina), MODIFIER OF SNC1, Protein, AI551343, deoxyribonucleic photolyase activity, Hydrochloride, Guanidine Sulfite (1:1), table salt, CES2A1, Vacuums, 1H-imidazol-3-ium, l(2)SH0323, SDCCAG33, ACETONITRILE, cyanomethane, CG13176, CYH1, Mass Spectrum, uree, photolyase activity, urea, Tripcellim, cerebral amyloid angiopathy, sample population, chromic formate, UREA, incised, Interleukin-1 beta convertase, Protein Gene Products, Trypure, concentration, wood spirit, 3.4.22.36, calcium salt, Mass., assay, SCH, hereditary cerebral haemorrhage with amyloidosis - Dutch type, sodium chloridesmall, Sumo, biological signaling, modifier, l(2)04493, Proteins, Gene, ATP Dependent Proteolysis Factor 1, Ubiquitin carboxyl extension protein 80, l(2)SH2 0182, Qualifier, CEP52, Smt3, SMT3, Human, SUMO, Ubiquitin A-52 residue ribosomal protein fusion product 1, reduced, CG4494, Ubiquitin-related 2, Protein, Gene Products, sumo, HMG-20, dSmt3, tiny, techniques, ATP-Dependent Proteolysis Factor 1, ubiquitin-like protein modifier, qualifier, Ubiq, disease qualifier, Ubiquitin, covalent modifier, High Mobility Protein 20, underdeveloped, Dm0342, dsmt3, Protein., hypoplasia, Human Ubiquitin, DmSmt3, Ubiquitin-related 1, proteins, procedures, DmSUMO-1, Modifier, signalling, Protein Gene Products, anon-EST:Posey240, APF-1, Gene Proteins, 40S ribosomal protein S27a, signalling process, 60S ribosomal protein L40, protein tag, signaling process, ubiquitin, Ubiquitin-related, disease characteristic, l(2)SH0182, protein tagging activity, Dmsmt3, DmelCG4494, single organism signaling, methodologyDIMETHYL SULFOXIDE, dmBest1, ion, Raw, DmelCG6264, Long Term, 5730420M11Rik, Polypeptides, protein polypeptide chains, M(3L)i, Drug Tolerance, Uba52, B1, Line, Software Engineering, establishment and maintenance of substrate location, Analysis, WMS, UBA52, AI194771, SET, Divorced, F, C79325, Analyses, M, phosphatase 2A inhibitor I2PP2A, dimethyl sulphoxide, Divorces, proteins, T6G21.3, Fast, DmelCG4299, set, RPL40, column, ACN, D1, Acn, sample, n, Homo sapiens disease, D9, Half Cystine, CEP80, M(3)RpS17, drug tolerance, FAST, Dimethylsulfoxid, SGS, LC-MS2, close to, DMSO, Stars, Data Set, establishment and maintenance of cellular component location., dmso, Zinc Cysteinate, immune system tolerance, acetylation, Longterm Effect, FASTK, CEP52, not genetically inherited, Spectrum Analysis, Software Tools, ACMICD, Computer Applications, (CH3)2SO, M(3)i(55), Computer Applications Software, TU15B, Ubiq, rpS17, Software Applications, MCO15.11, HLA-DR-associated protein II, DI-2, Source Software, I-2Dm, dbest1, proportionality, Spectrometry, pooled, MCO15_11, rate, inhibitor of granzyme A-activated DNase, RPS27A, human, 0610006J14Rik, experimental procedures, I-2PP1, SUMO 2, Applications, Self Tolerance, TAF-IBETA, liquid chromatography-tandem mass spectroscopy, TAF-Ibeta, STARS, Computer Software Applications, breadth, Effects, False, STK10, dimethylsulfoxyde, ATP Dependent Proteolysis Factor 1, precursor, protein-containing complex, body system, UBCEP2, Human, period, M(3)i, LC-MSMS, Gene Products, disease or disorder, system, Half-Cystine, Application, Open Source Softwares, sulfinylbis(methane), proportion, anatomical systems, Acinus, Longterm, Software Application, L Cysteine, VMD2, scattered, Open Source Software, 2pp2a, Long-Term, BMD, l(3)dtOA4, ions, man, CG10574, CD156, Computer Software Application, acinusL, Injectable, dimetil sulfoxido, 40S ribosomal protein S27a, 2PP2A, Tools, acinusS, M(3)i[55], dSET, dSet, peptidos, protein tagging activity, PTHB1, RP50, grupo, diffuse, Smt3b, Proteins, disorders, SUMO3, SMT3B, mKIAA0670, Cell, Engine, Tool, near to, polypeptide, SMALL UBIQUITIN-LIKE MODIFIER 2, Ubiquitin A-52 residue ribosomal protein fusion product 1, Immune Tolerance, native protein, I-2PP2A, C18, Ubiquitin-related 2, chemical analysis, Dm I-2, Long Term Effects, condition, BcDNA:RE44119, MFS1, background, Mass Spectrum Analyses, covalent modifier, Col4a-1, best, Separations, M(3)q, Striated muscle activator of Rho-dependent signaling, dimethyl sulfoxide, CD156a, anon-EST:Posey48, Longterm Effects, plan specification, Computer Programs, Gene Proteins, Applications Softwares, localisation, 60S ribosomal protein L40, protein tag, Smt3A, TI-225, vicinity of, M(3L)i[55], quotient, liquid chromatography tandem mass spectrometry, Ubiquitin-related, BEST, Data Analyses, Immunological Tolerance, liquid chromatography tandem mass spectroscopy, Rps17, Bru, IPP2A2, determination, RPS17, Gm1863, protein, peptide, M(3)67, peptido, ClvPrd, diseases, BANF, ARB, diseases and disorders, protein aggregate, Injectables, Effect, Computer Program, Mass Spectrum Analysis, Svc, human disease, LCMSMS, High Mobility Protein 20, peptides, reference sample, TAF-I, 2610036I19Rik, l(3)67BDo, Open, 2610510L13Rik, E430039A18Rik, min, Computer Programs and Programming, Ubiquitin-related 1, SCAN, free, SUMO-2, ATSUMO2, APF-1, IGAAD, fSAP152, DmelCG10574, ppm, GPHYSD2, Long-Term Effects, ratio, phapii, establishment and maintenance of substance location, CG6264, dimethyli sulfoxidum, Rps27a, methylsulfinylmethane, LC-MS/MS, M(3)67C, StF-IT-1, Search, HSMT3, Source Softwares, Programs, Spectroscopy, Program, single organism localization, Ionen, Computer Applications Softwares, Immunologic Tolerance, Softwares, Lines, dBest1, parent ion, Ubiquitin, (methanesulfinyl)methane, sumo-2, MS/MS, Rp S17, smt3h2, Human Ubiquitin, common, L-Cysteine, MASS, CG4299, organ system, small ubiquitin-like modifier 2, disease, SMT3H2, precursor ion, Tolerance, Polypeptide, i2pp2a, Proteomes, time, Gruppe, ubiquitin A-52 residue ribosomal protein fusion product 1, other disease, human being, Cysteine Hydrochloride, experimental, FBN, Gene, Smt3h2, ACINUS, Computer, Spectrum Analyses, Ubiquitin carboxyl extension protein 80, anon-WO0118547.380, PHAPII, LC-MS-MS, ubiquitin carboxyl extension protein 80, method, M(3)S33, template-activating factor I, polypeptide chain, Ms1, ECTOL1, Injection, method used in an experiment, Mass, establishment and maintenance of position, HMG-20, Del(8)44H, ubiquitin-like protein modifier, Separated, Mass Spectroscopy, S(O)Me2, SUM2, Search Engines, methods, MS1, experimental section, MS2, iones, ipp2a2, Acetylations, Minute, Ubb, Ubb2, Ubc, UBC, Fus1 protein, AL033289, non-neoplastic, OCTD, D8Ertd21e, DmelCG3922, 10^[-6], taf-ibeta, ATP:Fas-activated serine/threonine protein phosphotransferase activity, grupos, male sterility 1, hsmt3, ubiquitin, disorder, Long-Term Effect, Controlled, Applications Software, 2700054O04Rik, Open Source, data, Controlling, Computer Software, protein complex, igaad, mammalian, Cell Lines, total expressed protein, medical condition, Peptide, group, HUBCEP52, LC/MS/MS, Software Tool, sumo2, natural protein, Protein, I2PP2A, establishment and maintenance of localization, connected anatomical system, Dimethyl sulfoxide, ATP-Dependent Proteolysis Factor 1, Software, tandem MS, WMS2, Data Base, Mass Spectrum, Separation, Dbest, 10^[-9], L40, MALE STERILITY 1 PROTEIN, Engineering, Rest, single-organism localization, sample population, introduction, CG3922, Protein Gene Products, dSET/TAF-Ibeta, 2610030F17Rik, Ion, Data, approaches, SSKS, smt3b, Peptid, assay, variable, dimethyl sulfur oxide, SCH, AA407739, groupeprojections, Networks, multicellular organismal catabolic process, single-organism catabolic process, posttranslational modification, Kinship, Chromosome Segregations, DmelCG1258, SUMO-protein conjugation, Phases, protein, l(2)SH2 0182, phosphorylation, Smt3, SMT3, protein polypeptide chains, Mitotic M, Roles, KNL2, Knl2, Sumoylations, Smt3-protein conjugation, Concepts, Life Cycle, Analysis, protein aggregate, small ubiquitin-related protein 1 conjugation, present in fewer numbers in organism, 0973/08, Family Life Cycle, Mass Spectrum Analysis, Cell Division Cycles, posttranslational amino acid modification, Ubiquitylation, M Phases, High Mobility Protein 20, Kinship Network, Analyses, catabolism, DmMKLP1, C87313, hypoplasia, DmSmt3, Ubiquitin-related 1, proteins, protein sumolation, W, DmSUMO-1, anon-EST:Posey240, Ligase, APF-1, DNA-dependent, decreased, AU018689, PAV-KLP, papilla, Cell Cycles, Role Concepts, 26S proteasome, ubiquitination, 2610009E16Rik, Segregations, Family Life Cycles, Smt3p-protein conjugation, transcription from bacterial-type RNA polymerase promoter, PavKLP, Dmsmt3, close to, PAV, Pav, DmKLP1/pav, anatomical protrusion, wide/broad, DNA-dependent transcription, l(2)04493, acetylation, lamina, flanges, late, CEP52, Spectrum Analysis, results, Division Cycles, Spectroscopy, anon-EST:fe2A12, Role Concept, 2A12, KIF23, knsl5, shelf, MKLP1, Role, Filiation, CG1258, Ubiq, Ubiquitin, Mklp1, ParaT, proteasome, breakdown, PTM, Mitoses, Division Cycle, Knsl5, shelves, mKIAA1903, mklp-1, SLX5, Human Ubiquitin, Spectrometry, projection, ridge, organ system, Life Cycles, Ubiq., wide, Chromosome, cho1, spine, post-translational modification, bacterial transcription, Pav KLP, Mitotic, l(2)SH0182, CHO1, KNSL5, DmelCG4494, mklp1, Gruppe, sumoylation, transcription, Segregation, Family Member, SUMO-Conjugations, lamellae, Gene, Synthetases, ATP Dependent Proteolysis Factor 1, Network, Ubiquitin carboxyl extension protein 80, broad, Spectrum Analyses, protein-containing complex, process of organ, body system, Human, protrusion, lamella, method, reduced, polypeptide chain, PAV/KLP, subnumerary, method used in an experiment, Cycle, Gene Products, Mass, DNA Damage Response, HMG-20, system, pav-Klp, pav-KLP, dSmt3, tiny, ubiquitin-like protein modifier, Mass Spectroscopy, Cycles, HSA242977, anatomical systems, Research, Cell Division Cycle, cell-division cycle, Acetylations, Cell Division, ridges, posttranslational protein modification, decreased number, 40S ribosomal protein S27a, chromosome transmission, grupos, ubiquitin, MKLP-1, 0934/13, Kinship Networks, protein tagging activity, Family, laminae, Methylations, chromosome division, small, 3110001D19Rik, 6720403H11, Sumo, cellular transcription, grupo, Family Research, degradation, protein complex, anatomical process, Proteins, Phosphorylations, RGD1305497, Cell, group D, group, Concept, Family Members, near to, SUMO, Ubiquitin A-52 residue ribosomal protein fusion product 1, 2A12MEL, native protein, natural protein, CG4494, Ubiquitin-related 2, mitosis, Protein, Synthetase, sumo, Mitotic M Phase, anon2A12, methylation, ATP-Dependent Proteolysis Factor 1, connected anatomical system, SNURF, M Phase, flange, Mass Spectrum Analyses, organ process, C79407, covalent modifier, Mass Spectrum, Phase, underdeveloped, Dm0342, dsmt3, Pav-Klp, Pav-KLP, Gtrgeo8, Rest, DNA-templated, PAV-MKLP, M18BP1, post-translational amino acid modification, Protein Gene Products, plan specification, SUMO Conjugation, Gene Proteins, processes, process, 60S ribosomal protein L40, protein tag, Pav-klp, Families, approaches, vicinity of, Mitotic M Phases, processus, Ubiquitin-related, regulation, SUMO-Conjugation, Relatives, groupe, C14orf106projections, Networks, multicellular organismal catabolic process, single-organism catabolic process, posttranslational modification, Kinship, Chromosome Segregations, DmelCG1258, SUMO-protein conjugation, Phases, protein, l(2)SH2 0182, phosphorylation, Smt3, SMT3, protein polypeptide chains, Mitotic M, Roles, KNL2, Knl2, Sumoylations, Smt3-protein conjugation, Concepts, Life Cycle, Analysis, protein aggregate, small ubiquitin-related protein 1 conjugation, present in fewer numbers in organism, 0973/08, Family Life Cycle, Mass Spectrum Analysis, Cell Division Cycles, posttranslational amino acid modification, Ubiquitylation, M Phases, High Mobility Protein 20, Kinship Network, Analyses, catabolism, DmMKLP1, C87313, hypoplasia, DmSmt3, Ubiquitin-related 1, proteins, protein sumolation, W, DmSUMO-1, anon-EST:Posey240, Ligase, APF-1, DNA-dependent, decreased, AU018689, PAV-KLP, papilla, Cell Cycles, Role Concepts, 26S proteasome, ubiquitination, 2610009E16Rik, Segregations, Family Life Cycles, Smt3p-protein conjugation, transcription from bacterial-type RNA polymerase promoter, PavKLP, Dmsmt3, close to, PAV, Pav, DmKLP1/pav, anatomical protrusion, wide/broad, DNA-dependent transcription, l(2)04493, acetylation, lamina, flanges, late, CEP52, Spectrum Analysis, results, Division Cycles, Spectroscopy, anon-EST:fe2A12, Role Concept, 2A12, KIF23, knsl5, shelf, MKLP1, Role, Filiation, CG1258, Ubiq, Ubiquitin, Mklp1, ParaT, proteasome, breakdown, PTM, Mitoses, Division Cycle, Knsl5, shelves, mKIAA1903, mklp-1, SLX5, Human Ubiquitin, Spectrometry, projection, ridge, organ system, Life Cycles, Ubiq., wide, Chromosome, cho1, spine, post-translational modification, bacterial transcription, Pav KLP, Mitotic, l(2)SH0182, CHO1, KNSL5, DmelCG4494, mklp1, Gruppe, sumoylation, transcription, Segregation, Family Member, SUMO-Conjugations, lamellae, Gene, Synthetases, ATP Dependent Proteolysis Factor 1, Network, Ubiquitin carboxyl extension protein 80, broad, Spectrum Analyses, protein-containing complex, process of organ, body system, Human, protrusion, lamella, method, reduced, polypeptide chain, PAV/KLP, subnumerary, method used in an experiment, Cycle, Gene Products, Mass, DNA Damage Response, HMG-20, system, pav-Klp, pav-KLP, dSmt3, tiny, ubiquitin-like protein modifier, Mass Spectroscopy, Cycles, HSA242977, anatomical systems, Research, Cell Division Cycle, cell-division cycle, Acetylations, Cell Division, ridges, posttranslational protein modification, decreased number, 40S ribosomal protein S27a, chromosome transmission, grupos, ubiquitin, MKLP-1, 0934/13, Kinship Networks, protein tagging activity, Family, laminae, Methylations, chromosome division, small, 3110001D19Rik, 6720403H11, Sumo, cellular transcription, grupo, Family Research, degradation, protein complex, anatomical process, Proteins, Phosphorylations, RGD1305497, Cell, group D, group, Concept, Family Members, near to, SUMO, Ubiquitin A-52 residue ribosomal protein fusion product 1, 2A12MEL, native protein, natural protein, CG4494, Ubiquitin-related 2, mitosis, Protein, Synthetase, sumo, Mitotic M Phase, anon2A12, methylation, ATP-Dependent Proteolysis Factor 1, connected anatomical system, SNURF, M Phase, flange, Mass Spectrum Analyses, organ process, C79407, covalent modifier, Mass Spectrum, Phase, underdeveloped, Dm0342, dsmt3, Pav-Klp, Pav-KLP, Gtrgeo8, Rest, DNA-templated, PAV-MKLP, M18BP1, post-translational amino acid modification, Protein Gene Products, plan specification, SUMO Conjugation, Gene Proteins, processes, process, 60S ribosomal protein L40, protein tag, Pav-klp, Families, approaches, vicinity of, Mitotic M Phases, processus, Ubiquitin-related, regulation, SUMO-Conjugation, Relatives, groupe, C14orf106Sumo, biological signaling, l(2)04493, Proteins, Gene, ATP Dependent Proteolysis Factor 1, l(2)SH2 0182, Ubiquitin carboxyl extension protein 80, CEP52, Smt3, SMT3, Human, SUMO, Ubiquitin A-52 residue ribosomal protein fusion product 1, CG4494, Ubiquitin-related 2, Protein, Gene Products, sumo, HMG-20, dSmt3, techniques, ATP-Dependent Proteolysis Factor 1, ubiquitin-like protein modifier, Ubiq, Ubiquitin, covalent modifier, High Mobility Protein 20, Dm0342, dsmt3, Protein., DmSmt3, Human Ubiquitin, Ubiquitin-related 1, proteins, procedures, DmSUMO-1, signalling, Protein Gene Products, anon-EST:Posey240, APF-1, Gene Proteins, 40S ribosomal protein S27a, signalling process, 60S ribosomal protein L40, protein tag, signaling process, ubiquitin, Ubiquitin-related, l(2)SH0182, protein tagging activity, Dmsmt3, DmelCG4494, single organism signaling, methodology392trueConverging SUMO and ubiquitin signaling: improved methodology identifies co-modified target proteinsPost translational protein modifications (PTMs) including small chemical groups and small proteins, belonging to the ubiquitin family, are essential for virtually all cellular processes. In addition to modification by a single PTM, proteins can be modified by a combination of different modifiers, which are able to influence each other. Since little is known about crosstalk between different ubiquitin family members, we developed an improved method enabling identification of co-modified proteins on a system-wide level using mass spectrometry. We focused on the role of crosstalk between SUMO and ubiquitin during proteasomal degradation. Using two complementary approaches, we identified 498 proteins to be significantly co-modified by SUMO and ubiquitin upon MG132 treatment. These targets included many enzymatic components of PTM machinery, involved in SUMOylation and ubiquitylation, but also phosphorylation, methylation and acetylation, revealing a highly complex interconnected network of crosstalk between different PTMs. In addition various other biological processes were found to be significantly enriched within the group of co-modified proteins, including transcription, DNA repair and the cell cycle. Interestingly, the latter group mostly consisted of proteins involved in mitosis, including a subset of chromosome segregation regulators. We hypothesize that group modification by SUMO-targeted ubiquitin ligases regulates the stability of the identified subset of mitotic proteins, which ensures proper chromosome segregation. The mitotic regulators KIF23 and MIS18BP1 were verified to be co-modified by SUMO and ubiquitin upon inhibition of the proteasome and subsequently identified as novel RNF4 targets. Both modifications on MIS18BP1 were observed to increase simultaneously during late mitosis, while the total protein level decreased immediately afterwards. These results confirm the regulation of MIS18BP1 via SUMO-ubiquitin crosstalk during mitosis. Combined, our work highlights extensive crosstalk between SUMO and ubiquitin, providing a resource for further unraveling of SUMO-ubiquitin crosstalk.2017-09-292017-09-13PXD007733280655164609918049851114542249215684354407128421832704236333635119834300663636275838569636312176666888811143015557114526314526236493461312695736664923916193641106656889511363647627236611123294422295558323231225863654346079251936591432059712938655819125023211957748186619682158672256807617554373592536684081691804542787751201089225058367414795641003923694204943344597414200129352936715675936438677160540483131528269304562716391624292042779447623763696121810032962922113763948840479NCBITaxon:21571315273105106720596872016516368407173630969893610478214836432042106132049138169374327732046453511110321745921612928867128867641006945694101676341021076127735113165821358430141729817463367541287356756840301350621813019411192943936711041136445913949843202530127014987511047594120478861347495153721109716529982790513980317585439801260866398081375755518320196447111415542193255521138343911931819813981810269701069955529NCBITaxon:47518502983354764146547798251021691572739823897648407413735186287054824153NCBITaxon:50557984467525100036433414754803597413708115466547898353221599838106771034819852348780422531488414222863479461571871599191572952929211752719860148840633424951869123423515233598589859886559746499870392021110723635272159874190828743418235024145113969293190259390719017463601237474636172262536001319722119158531058859698951105198997237989772000263419610264266722758858156899628737562690724420237172019998570724074767927459615333554319313355418400513686150596866628666134751536329NCBITaxon:17632862873488376705802718481865368595986075105135294296110098402321000734882412336363071047005187878945556449447NCBITaxon:13356265419591399878168119011478281055524NCBITaxon:96061358584690081979153519307285930085264114090051184995262254121337591390143811139442370416744312044143708351581990119519532590841370137023943222774862615835583715371633376057267998086371137121906503714759172073127262981274575727090309031372814243371421044792491108239412NCBITaxon:961514780269266100658137352750454135663816475203750644223669502129349790546322455525365199764990814389923745374726473237602647312887055955362527403271602938511497864794362938247943722511775299167387193844137659393432715910022637674234442345467001287689591198467041464792936528034280382803510803481793922950271131681911265269971028621076015973611431140981087135547743658142450722953379698204232124223321803369822347081645733165467599287770203829314125295508114817448886437452649997283035718308550717629970767892683704151562887651388563924561152086595551655181135491432137159749535026143213858781707683194963401614280053164610466038151845474485009747697477118051240174841855601555479204274201488711053253297261005941289093164359900138943828354232423310829341220585604162991311227317018799154291312678181871049316407299467303337330485264638301130641399253064236121162065261610337286129940207340115357652611114716133865431201725323712552329935316317NCBITaxon:11084993841063267872862321933486208621160791392227681196125522814953932132232334774121827532917573129948861814084341033519963139248811533811533933043937556992862633911209488624157183997586444237486435332444260665332413439097682127178637863999839986865535291486541042986357358257632884019864982380102868899958665220341632138119380625631886571303443112130886581144889198466645463NCBITaxon:11072NCBITaxon:11071142800628835374375116597145943116598854331116781077854314408043933434NCBITaxon:11082693977382189931824431935128387145953116885311213278695berry86941003335869724436647015011142869215047514991054842047147251208470146497444974311700893062186441930611076323308056603960162179748258539460553965132127905688133775129817660171121478610502366946532051124501897441739412450128853183889198679488531911137910371647351660152943631631198573896939343237956026087121346334323223544314275241433611334565560291699636436802790105603140818838185733703401881274414437552162038713985584081724055986091838272759212744234081702759812744201274426235455382117627594328247480119915095648188229120455148167238473848190695148498101510693731263753405203860186497405224052141856451804NCBITaxon:8134780091522754847938703338499172261756109461614351203258120550523525944732054938646512138803766861206781122022307418670511867057387334869121938791868023980071868012154514683514081861508471206777691838851608611388869152167786721279010087043512317196928123929486771332261262198131882682411229265716329576938124712461485451328388348392949113903631204162665890771514880036942694169436945187901015080817053942065421055687317447127015329242814111946691169601454586953201019812646905505371408157697392830312383911283128212802811010832825253531522811235315349825741948153623142410145481NCBInr 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