Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/H082758_uPAC_10_PRC-4563_BodineLab_GlyGly_Ctrl-5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/F000468_2p_uPAC7_PRC-4875_BodineLab_Shotgun_Control_rep_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/H082752_uPAC_10_PRC-4563_BodineLab_GlyGly_Murf-3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/H082749_uPAC_10_PRC-4563_BodineLab_GlyGly_Ctrl-3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/F000462_2p_uPAC7_PRC-4875_BodineLab_Shotgun_MuRF1_rep_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/F000454_2p_uPAC8_PRC-4875_BodineLab_Shotgun_Control_rep_4_20190712202135.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/H082747_uPAC_10_PRC-4563_BodineLab_GlyGly_Murf-2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/F000452_2p_uPAC7_PRC-4875_BodineLab_Shotgun_MuRF1_rep_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/H082760_uPAC_10_PRC-4563_BodineLab_GlyGly_Murf-5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/H082754_uPAC_10_PRC-4563_BodineLab_GlyGly_Ctrl-4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/H082756_uPAC_10_PRC-4563_BodineLab_GlyGly_Murf-4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/F000458_2p_uPAC7_PRC-4875_BodineLab_Shotgun_Control_rep_5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/F000466_2p_uPAC7_PRC-4875_BodineLab_Shotgun_MuRF1_rep_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/F000460_2p_uPAC7_PRC-4875_BodineLab_Shotgun_MuRF1_rep_5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/F000456_2p_uPAC7_PRC-4875_BodineLab_Shotgun_Control_rep_2_20190713014123.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/H082745_uPAC_10_PRC-4563_BodineLab_GlyGly_Ctrl-2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/txt_PTM.7zftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/07/PXD023872/txt_SG.7zprimaryOK200sue-bodine@uiowa.eduDelphi Van HaverMass SpectrometryShotgun proteomicsAndromedaMaxQuantProlineMuscle atrophyProtein degradationMurf1ElectroporationUbiquitin proteomicshttp://www.ebi.ac.uk/pride/archive/projects/PXD023872Tibialis Anterior4 C57BL/6 mice were electroporated with the untagged MuRF1 plasmid in one TA muscle and an empty vector (control) plasmid in the contralateral TA muscle. After 14 days, the mice were anesthetized with isoflurane, and the TA muscles were excised, weighed, and frozen in liquid nitrogen. Muscle tissues were homogenized in 5 ml urea lysis buffer (9 M urea, 20 mM HEPES pH 8.0). The samples were sonicated with 3 pulses of 15 s at an amplitude of 20% using a 3 mm probe, with incubation on ice for 1 min between pulses. After centrifugation for 15 min at 20,000xg at room temperature (RT), proteins were reduced with 5 mM DTT and incubation for 30 min at 55˚C, then alkylated with 10 mM CAA and incubation for 15 min at RT in the dark. The protein concentration was measured using a Bradford assay (Bio-rad) and from each sample 13.8 mg protein was used to continue the protocol. Samples were diluted with 20 mM HEPES pH 8.0 to 4 M urea and proteins were digested with 69 µg LysC (Wako) (1/200, w/w) for 4 hours at 37°C. Samples were diluted to 2 M urea and digested with 69 µg trypsin (Promega) (1/200, w/w) overnight at 37˚C. The resulting peptide mixture was acidified with trifluoroacetic acid (TFA) and after 15 min incubation on ice, samples were centrifuged for 15 min at 1,780xg at RT. Immunocapture of GlyGly-modified peptides was then performed using the PTMScan® Ubiquitin Remnant Motif (K-ε-GG) Kit (Cell Signaling Technology) according to the manufacturer’s instructions. Before enrichment, aliquots corresponding to 200 µg of digested protein were taken for shotgun (SG) proteomics analysis. Purified di-glycine (GG) modified peptides were dried under vacuum in HPLC inserts, and stored at −20 °C until LC-MS/MS analysis. Purified peptides for SG analysis were re-dissolved in 20 µl solvent A (0.1% TFA in water/ACN (98:2, v/v) and 2 µg of each sample was injected for LC-MS/MS analysis on an Ultimate 3000 RSLCnano system in-line connected to an Orbitrap Fusion Lumos MS (Thermo) equipped with a pneu-Nimbus dual ion source (Phoenix S&T). Trapping was performed at 10 μl/min for 4 min in solvent A on a 20 mm trapping column (made in-house, 100 μm internal diameter (I.D.), 5 μm beads, C18 Reprosil-HD, Dr. Maisch, Germany) and the sample was loaded on a 200 cm long micro pillar array column (PharmaFluidics) with C18-endcapped functionality mounted in the Ultimate 3000’s column oven at 50°C. For proper ionization, a fused silica PicoTip emitter (10 µm I.D., New Objective) was connected to the µPAC™ outlet union and a grounded connection was provided to this union. Peptides were eluted by a nonlinear increase from 1 to 55% MS solvent B (0.1% FA in water/ACN (2:8, v/v)) over 145 min, first at a flow rate of 750 nl/min, then at 300 nl/min, followed by a 10-min wash reaching 99% MS solvent B and re-equilibration with MS solvent A (0.1% FA in water). The MS was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition. Full-scan MS spectra (300-1500 m/z) were acquired in 3 s acquisition cycles at a resolution of 1.2E5 in the Orbitrap analyzer after accumulation to a target AGC value of 2E5 with a maximum injection time of 30 ms. The precursor ions were filtered for charge states (2-7 required), dynamic range (60 s; +/- 10 ppm window) and intensity (minimal intensity of 3E4). The precursor ions were selected in the multipole with an isolation window of 1.2 Da and accumulated to an AGC target of 5E3 or a maximum injection time of 40 ms and activated using HCD fragmentation (34% NCE). The fragments were analyzed in the Ion Trap Analyzer at normal scan rate. Purified GG modified peptides were re-dissolved in 20 µl solvent A of which 15 µl was injected for LC-MS/MS analysis on an Ultimate 3000 RSLCnano system in-line connected to a Q Exactive HF MS (Thermo). Trapping and loading the sample was performed as described for the SG analysis. Peptides were eluted by a nonlinear increase from 1 to 55% MS solvent B over 116 min, first at a flow rate of 750 nl/min, then at 300 nl/min, followed by a 14-min wash reaching 99% MS solvent B and re-equilibration with MS solvent A. The MS was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the 8 most abundant ion peaks per MS spectrum. Full-scan MS spectra (375-1500 m/z) were acquired at a resolution of 6E4 in the orbitrap analyser after accumulation to a target value of 3E6. The 8 most intense ions above a threshold value of 8.3E3 were isolated (window of 1.5 Th) for fragmentation at a normalized collision energy of 28% after filling the trap at a target value of 1E5 for maximum 120 ms. MS/MS spectra (200-2000 m/z) were acquired at a resolution of 1.5E4 in the orbitrap analyser. The S-lens RF level was set at 50 and we excluded precursor ions with single, unassigned and >7 charge states from fragmentation selection. QCloud was used to control instrument longitudinal performance during the project.PrideMS1 intensity based label-free quantification methodubiquitination signature dipeptidyl lysineiodoacetamide derivatized residuemonohydroxylated residueacetylated residueData analysis was performed with MaxQuant (version 1.6.3.4) using the Andromeda search engine with default search settings including a false discovery rate set at 1% on the peptide and protein level. Two different searches were performed to analyze the spectra from the di-glycine-enriched samples and the SG samples. In both searches, spectra were interrogated against the mouse proteins in the Swiss-Prot Reference Proteome database (database release version of June 2019 containing 22,282 mouse protein sequences, (http://www.uniprot.org)). The mass tolerance for precursor and fragment ions was set to 4.5 and 20 ppm, respectively, during the main search. Enzyme specificity was set as C-terminal to arginine and lysine, also allowing cleavage at proline bonds with a maximum of three missed cleavages. Variable modifications were set to oxidation of methionine residues, acetylation of protein N-termini and GlyGly modification of lysine residues, while carbamidomethylation of cysteine residues was set as fixed modification. Matching between runs was enabled with a matching time window of 0.7 min and an alignment time window of 20 min. Only proteins with at least one unique or razor peptide were retained leading to the identification of 1,412 proteins and 13,329 GlyGly modified sites. Proteins were quantified by the MaxLFQ algorithm integrated in the MaxQuant software. A minimum ratio count of two unique or razor peptides was required for quantification. Further data analysis of the SG results was performed with the Perseus software (version 1.6.2.1) after loading the proteingroups file from MaxQuant. Reverse database hits were removed, LFQ intensities were log2 transformed and replicate samples were grouped. Proteins with less than three valid values in at least one group were removed and missing values were imputed from a normal distribution around the detection limit leading to a list of 844 quantified proteins that was used for further data analysis. Then, a t-test was performed (FDR=0.05 and s0=1) to compare control and MuRF1 samples and a volcano plot was generated. 24 proteins were found to be significantly regulated. For the analysis of the di-glycine modified peptide data, the GlyGly(K)Sites file was loaded in the Perseus software (version 1.6.2.1). Reverse hits were removed, the site table was expanded, the intensity values were log2 transformed and the median was subtracted. Replicate samples were grouped, GlyGly(K)sites with less than three valid values in at least one group were removed and missing values were imputed from a normal distribution around the detection limit leading to a list of 963 quantified GlyGly peptides that was used for further data analysis. Then, a t-test was performed (FDR=0.05 and s0=1) to compare control and MuRF1 samples and a volcano plot was generated. 169 GlyGly peptides were significantly regulated and plotted in a heatmap after non-supervised hierarchical clustering. GO term enrichment analyses were performed using DAVID with a 0.1 EASE score cutoff.ProteomicsSue BodineOrbitrap Fusion LumosQ Exactive HFDepartment of Internal Medicine, Division of Endocrinology and Metabolism, Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242 USAPARTIALMus Musculus (mouse)delphi.vanhaver@vib-ugent.be34179788 Baehr LM, Hughes DC, Lynch SA, Van Haver D, Maia TM, Marshall AG, Radoshevich L, Impens F, Waddell DS, Bodine SC. Identification of the MuRF1 Skeletal Muscle Ubiquitylome Through Quantitative Proteomics. Function (Oxf). 2021 2(4):zqab029 10.1093/function/zqab029VIB Proteomics CoreBelgiumMuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine whether MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.Identification of the MuRF1 Skeletal Muscle Ubiquitylome Through Quantitative Proteomics.Baehr Leslie M LM, Hughes David C DC, Lynch Sarah A SA, Van Haver Delphi D, Maia Teresa Mendes TM, Marshall Andrea G AG, Radoshevich Lilliana L, Impens Francis F, Waddell David S DS, Bodine Sue C SCisofluranum, IL1BC, ion, Laboratory, TFA, Aminoessigsaeure, CASP-1, rad, Gly, Basodexan, Apaf-1, House Mouse, Solvent, Long Term, RP11-508D10.1, GLYCINE, 5730420M11Rik, Trifluoroacetate, dmTAF[[II]]230, lenses, Polypeptides, protein polypeptide chains, KL receptor activity, B1, SCO5, 1, Urea, 2, SCO1, Il1bc, Gsfsow3, IRF, 1-trifluoro-, average, SET, type 5 acid phosphatase, G, C79325, hac-1, TFIID TAF250, cel, Glycine, DmelCG13176, N, weight-weight percentage, phosphatase 2A inhibitor I2PP2A, Tissue, Salt, ocular lens, ORF19, proteins, W, IL-1BC, HPLC, AI047805, DmelCG4299, isolation and purification, set, T-cell antigen Gp39, decreased, column, Glycocoll, scientific observation, ACN, sample, Acn, D1, Bs, fused to, autolysin activity, TNFSF5, RF1, single organism signaling, accumulated, LC-MS2, Cobalt Salt Glycine, dTAF[[II]]230, Caspase-1 subunit p10, Iris RING finger protein, Glycine Phosphate, 6.3.2.-, Tnfrsf5, familial, anon-53Fa, TAF200, Longterm Effect, set of skeletal muscles, l(2)SH0173, Monosodium Salt Glycine, Normalcy, CEP52, not genetically inherited, Aerrane, Copper Salt Glycine, DmelCG7788, hCD40L, PBT, Stickstoff, Dm1, DmelCG42628, IMD3, Raps, 1110049F14Rik, CG6829, Ubiq, Hydrogen Oxide, dark/hac-1/dapaf-1, Acid, reticulate acropigmentation of Dohi, pbt, Industrial, Industrial Arts, HLA-DR-associated protein II, Caspase-14 subunit p10, DI-2, Glycine Sulfate (3:1), lens, I-2Dm, FU, Swiss Mouse, NKTL, dyschromatosis symmetrica hereditaria 1, Dmel_CG15720, pooled, Caspase-14 subunit p19, Cobalt Salt, inhibitor of granzyme A-activated DNase, study protocol, beta Trypsin, w/w, I-2PP1, nitrogen, TAF-IBETA, Taf250, liquid chromatography-tandem mass spectroscopy, Drice, Fu, bacteriolytic toxin activity, Mouse, TAF-Ibeta, fused, DRICE, TAF230, krk1, FUSED, 3.4.22.-, knobbly, Peptidomics, Effects, Ly62, DrIce, DrICE, TAPK, Fused, muscles, Lens <eudicots>, dyschromatosis symmetrica hereditaria, mini-ICE, glycine, Carbamide, ATP Dependent Proteolysis Factor 1, precursor, protein-containing complex, thomson, TR-AP, body system, DmelCG5692, CG7826, lens crystallina, Human, mass-to-charge ratio, period, 1-chloro-2, isolation, IT, LC-MSMS, GP39, CG7835, Gene Products, Mus musculus domesticus, CG42273, system, Carmol, gp39, Normalities, CD154, Cesium Trifluoroacetate, anatomical systems, dTAF[[II]]250, FAM39E, GKLP, CATC4, pins, Acinus, Forane, Longterm, Tissues, cell, Monosodium Salt, PCE-2, beta-Trypsin, 2pp2a, Long-Term, ions, study assay, PINS, MuRF-1, 2-7, CG10574, acinusL, Injectable, dTAF250, 40S ribosomal protein S27a, PCTAIRE2-binding protein, MGC:45012, 2PP2A, Caspase-1 subunit p20, acinusS, dSET, dSet, peptidos, Kb, protein tagging activity, 4-(2-hydroxyethyl)-, PTHB1, Ki, measuring, dark/dapaf-1/hac-1, proto-oncogene c-Kit, NTKL, mass percentage, lens capsule, 2-chloro-2-(difluoromethoxy)-1, CG5692, caspase 3, polypeptide motif, Monopotassium Salt Glycine, dApaf-1, Proteins, Copper Salt, BG:DS00004.13, CE-2, DSH, DSH1, Mischung, ACP5, buffer, motif, SPENCDI, ribosome-associated ubiquitin-dependent protein breakdown, Cell, mKIAA0670, B930045J24, dTAF230, CD142, polypeptide, RING finger protein 28, APAF1, Wash2, Wash1, Ubiquitin A-52 residue ribosomal protein fusion product 1, Temperatures, native protein, carbonyldiamide, Hac-1, I-2PP2A, Ubiquitin-related 2, KIT ligand receptor activity, C18, chemical analysis, Long Term Effects, Dm I-2, MURF1, TAF[[II]]250/230, MURF2, Pins, XKrk1, AU020952, Taf[[II]]250, covalent modifier, MuRF1, Murf1, muscles set, holin, underdeveloped, Dark/Dapaf-1/HAC1, House Mice, Monopotassium Salt, IL-1 beta-converting enzyme, muscle, azote, Murf, Interleukin-1 beta-converting enzyme, CD117, Glyzin, signalling, Longterm Effects, ME-IV, plan specification, Gene Proteins, Rnf28, signalling process, 60S ribosomal protein L40, C-Kit, protein tag, Ssm, Aminoacetic, Glykokoll, Ethane, Isoflurane, CG4346, xkl-1, liquid chromatography tandem mass spectrometry, Ubiquitin-related, aminoacetic acid, TRACP, RNF28, Muscle Tissue, Calcium Salt, liquid chromatography tandem mass spectroscopy, Hac-1/Dark, SMRZ, Glycine Carbonate (2:1), IPP2A2, muscle system, Calcium Salt Glycine, determination, H2N-CH2-COOH, selection process, DmelCG6829, Mus domesticus, 2-trifluoroethyl difluoromethyl ether, instrument configuration, CG7788, crice, HEPES Monosodium Salt, CASP-14, protein, Xkl-1, CG15720, Monoammonium, lysis, Monosodium, HCHWA, peptide, Tnfsf5, Gsfsco1, ClvPrd, peptido, BANF, Calcium Salt (2:1), kinky, Monoammonium Salt Glycine, Min, Apaf1, protein aggregate, present in fewer numbers in organism, ARK, Gsfsco5, Injectables, Effect, DmelCG42273, SOW3, T-BAM, HEPES Monosodium, Muscle Tissues, rsh, LCMSMS, tartrate-resistant acid ATPase, High Mobility Protein 20, reference sample, peptides, TAF-I, E927b, long, 2610036I19Rik, 2610510L13Rik, hypoplasia, min, arc, crystalline lens, Ubiquitin-related 1, Swiss Mice, mAPC, Phosphate, analyzer, purification, Forene, ark, SCAN, T1, genetic, APF-1, TRAP3, IGAAD, fSAP152, Tudor repeat associator with PCTAIRE-2, 1-Piperazineethanesulfonic acid, DmelCG10574, ICE, signaling process, ppm, trifluoro-, AI316800, TF, necrosis, dApaf1, T5ap, Sl, MGC - 45012, Long-Term Effects, Dmel_CG4346, symmetric dyschromatosis of the extremities, CD40L, phapii, ice, iCE, D-Apaf-1, drice, set of muscles, Arts, mouse, LC-MS/MS, Cd40l, Dmel_CG7826, StF-IT-1, Tr-kit, Th, TAFII-250, TAF250/230, aminoethanoic acid, Muscle RING finger protein 1, ur, hereditary cerebral hemorrhage with amyloidosis - Dutch type, any method, carbamide, TAFII250, HCD, Ionen, drIce, drICE, Mini-ICE, kl1-A, bacteriocin activity, TNF-related activation protein, KIT, WASH, Ly-62, Dmel_CG7835, Acetic acid, Mnb, MNB, Ubiquitin, parent ion, tyrosine-protein kinase Kit, Mus musculus, mice, ribosome-associated degradation, Hgly, P45, familial reticulate acropigmentation of Dohi, Trifluoroacetic, Human Ubiquitin, AW743063, ribosome-associated ubiquitin-dependent protein degradation, isoflurano, hac1, kit, CG4299, AW124434, AI326936, MICE, CG17603, merged with, TAF[[II]], isoflurane, 2-chloro-2-difluoromethoxy-1, muscle element, domesticus, REM3, organ system, dapaf-1S, H2NC(O)NH2, apaf-1, Health, lysin activity, dapaf-1L, precursor ion, Cesium, SR3-5, TrATPase, IGM, p45, microarray, CD40LG, Polypeptide, HEPES, 7N, RAD1, i2pp2a, 1728, time, Lens <bivalves>, d230, biological signaling, dutch hereditary cerebral amyloid angiopathy, Sodium Hydrogen Carbonate, Dapaf-1/HAC-1, TRAP, p50, 1-trifluoroethane, musculature system, Monopotassium, SCF receptor activity, rapsyn, Gene, dTAFII250, Ubiquitin carboxyl extension protein 80, ACINUS, EfW1, PHAPII, LC-MS-MS, Buffer, Dark/Hac-1/dApaf1, Karbamid, method, Hac1, Dark/Hac-1/dApaf-1, polypeptide chain, reduced, scfr, template-activating factor I, House, dmTAF1, subnumerary, Taf230, Injection, p63, method used in an experiment, Harnstoff, p65, HMG-20, camera-type eye lens, tiny, Glycine Hydrochloride (2:1), ubiquitin-like protein modifier, CD40-L, Mice, SCFR, Pulses, Pro-Mega, TAF250, Fdc, Taf200, Glycine Hydrochloride, Swiss, iones, ipp2a2, dapaf-1, Taf1p, dapaf, dark, instrument., decreased number, Salt Glycine, Glycin, 10^[-6], DARK, TEIF, Trap, taf-ibeta, musculus, ubiquitin, ribosome-associated ubiquitin-dependent protein catabolism, HIGM1, Leimzucker, liquid, N-2-Hydroxyethylpiperazine-N'-2'-ethanesulfonic Acid, 2-aminoacetic acid, Long-Term Effect, mKIAA1278, dArk, TAF, Dapaf-1, eye lens, C78062, Controlled, small, Monoammonium Salt, P105, Controlling, DYRK1, data, 5E4, TAF[[II]]250, Glycine Phosphate (1:1), CG42628, musculi, protein complex, PCTAIRE2BP, TSH1, Aminoacetic acid, igaad, l(3)84Ab, apaf1, Bp50, acropigmentation of Dohi, Striated muscle RING zinc finger protein, Muscle, NY-CO-33, Peptide, CAA, Axin, Tripartite motif-containing protein 63, LC/MS/MS, Glycine Carbonate (1:1), natural protein, Mus, p230, Protein, I2PP2A, Hydrochloride, TFIID, Dyrk1, CES2A1, Dark, Vacuums, ATP-Dependent Proteolysis Factor 1, connected anatomical system, SDCCAG33, Dark/Apaf-I, CG13176, Aminoacetic Acid, c-KIT, TAF[[II]]230, uree, Normality, urea, CC1, Tripcellim, N 2 Hydroxyethylpiperazine N' 2' ethanesulfonic Acid, cerebral amyloid angiopathy, TAF[II]250, Monolithium Salt, dApaf-1/DARK/HAC-1, sample population, UREA, Laboratory Mice, nitrogeno, Interleukin-1 beta convertase, Protein Gene Products, Muscle-specific RING finger protein 1, Trypure, dSET/TAF-Ibeta, c-kit, 2610030F17Rik, DmelCG17603, Ion, joined with, 3.4.22.36, musculature, Peptid, RAD, Rad, assay, SCH, AA407739, 1700112N14Rik, Laboratory Mouse, hereditary cerebral haemorrhage with amyloidosis - Dutch type, coalesced, dAPAF-1, muscle group, RCB1376, TAF1Peptidomics., SMRZ, MuRF1, Murf1, Iris RING finger protein, 6.3.2.-, number, Striated muscle RING zinc finger protein, Murf, Muscle RING finger protein 1, MuRF-1, skeletal muscle, presence, Muscle-specific RING finger protein 1, RING finger protein 28, Tripartite motif-containing protein 63, somatic muscle, count in organism, Rnf28, MURF1, MURF2, quantitative, RNF28, RF1, IRF, striated muscle, skeletal muscle system, presence or absence in organismScx, ion, Lysine Hydrochloride, Aminoessigsaeure, Gly, Kap-alpha2, Long Term, ter, 5730420M11Rik, GLYCINE, Polypeptides, protein polypeptide chains, Lysine Acetate, L-Proline, 2, Software Engineering, Analysis, WMS, IRF, L Lysine, SET, (S)-2-amino-5-guanidinopentanoic acid, F, G, Analyses, K, Glycine, M, phosphatase 2A inhibitor I2PP2A, R, Dimp-alpha2, Arginine, proteins, Monohydrate DL-Arginine Acetate, DmelCG4299, AI047805, set, LYS, Glycocoll, Bsg75C, Lysin, Half Cystine, RF1, SGS, NK/GPI, Gpi, Cobalt Salt Glycine, Iris RING finger protein, Glycine Phosphate, Zinc Cysteinate, 6.3.2.-, alphaKap2, acetylation, Longterm Effect, arginine, Monosodium Salt Glycine, IMPalpha2, Copper Salt Glycine, Software Tools, Expanded, ACMICD, Computer Applications, count, Dm1, Algorithm, 1270, Computer Applications Software, Distributions, Methionin, NK|GPI, methionine, Hmet, anon-WO0140519.258, Acid, Software Applications, HLA-DR-associated protein II, DI-2, Glycine Sulfate (3:1), Source Software, I-2Dm, proportionality, expanded, Ex, rate, Cobalt Salt, alpha, inhibitor of granzyme A-activated DNase, oho31, I-2PP1, Applications, TAF-IBETA, TAF-Ibeta, Mouse, Liquimeth, Bglap-rs1, Computer Software Applications, big, 2-amino-4-(methylsulfanyl)butanoic acid, False, Effects, region or site annotation, CG4799, glycine, Antp P1, precursor, pen, Antp P2, protein-containing complex, CG7826, importin alpha2/pendulin, period, large, Gaussian Distribution, DmelCG4114, Hu, Gene Products, CG7835, CG42273, l(2)k14401, Pgi, Lysine, Half-Cystine, Distribution, alpha2, 3.4, Application, Open Source Softwares, proportion, positional, Longterm, Gpi-1r, Gpi-1s, L Cysteine, Software Application, Phi, Monosodium Salt, Gpi-1t, Open Source Software, 2pp2a, Long-Term, ions, MuRF-1, CG1028, CG10574, Computer Software Application, DmelCG4216, 2PP2A, 2-amino-4-(methylthio)butanoic acid, Tools, L Proline, Kpna2, dSET, dSet, L-Arg, peptidos, 2.1, metionina, grupo, Monopotassium Salt Glycine, Proteins, CG4114, Copper Salt, MF, DmAntp, L-Isomer, Amf, Engine, Tool, polypeptide, RING finger protein 28, DmelCG4799, native protein, I-2PP2A, L-Isomer Arginine, Acetate, Dm I-2, Long Term Effects, chemical analysis, MURF1, MURF2, MFS1, L-Lysine, NK, Met, AU020952, Ant, MuRF1, Murf1, (2S)-2-amino-5-guanidinopentanoic acid, CG4216, Kap alpha2, Monopotassium Salt, Murf, Glyzin, Longterm Effects, ME-IV, l(2)01270, Computer Programs, Gene Proteins, Ns, Applications Softwares, Rnf28, DmelCG1028, binding_or_interaction_site, Aminoacetic, Glykokoll, Term., quotient, DRO15DC96Z, aminoacetic acid, RNF28, Enisyl, Data Analyses, Calcium Salt, IPP2A2, Glycine Carbonate (2:1), SMRZ, ANT-C, Calcium Salt Glycine, H2N-CH2-COOH, determination, L-arginine, DL-Arginine Acetate, Antp1, OHO31, cleavage, Monohydrate, protein, Monoammonium, Monosodium, brl, Normal Distributions, binding site, peptide, L-Isomer Methionine, Methionine, pen-2, ClvPrd, peptido, Calcium Salt (2:1), AntP1, Monoammonium Salt Glycine, Min, Arg, protein aggregate, Gpi-1, impalpha2, Effect, Computer Program, DmelCG42273, l(2)144/1, peptides, reference sample, TAF-I, Nlk, Open, (S)-2-Amino-5-guanidinovaleric acid, epsilon-diaminocaproic acid, min, Computer Programs and Programming, mAPC, Phosphate, imp-alpha2, BG:DS07700.1, IGAAD, DmelCG10574, ppm, Normal, ANT-P, house mouse, GPHYSD2, Racemethionine, Long-Term Effects, ratio, L-Arginin, phapii, oho-31, bs29g06.y1, mouse, Arginine Hydrochloride, 2-Amino-4-(methylthio)butyric acid, Dmel_CG7826, StF-IT-1, Search, aminoethanoic acid, Muscle RING finger protein 1, Source Softwares, results, Aus, Programs, Program, mOC-X, Ionen, lysine, Computer Applications Softwares, median, Softwares, Dmel_CG7835, region, parent ion, L-Arginine, Mnb, MNB, ORG, Org, positional polypeptide feature, mice, Hgly, Gpi1-r, Gpi1-s, L-Cysteine, CG4299, MASS, AW124434, Gpi1-t, precursor ion, enlarged, l(2)ey, Polypeptide, i2pp2a, Gaussian, Proteomes, time, Rch1, Gpi1s, Oho31, Gruppe, Cysteine Hydrochloride, Sodium Hydrogen Carbonate, AI461847, Dalpha2, Monopotassium, L Isomer, DMANTPE1, FBN, Gene, Computer, PHAPII, L-(+)-arginine, template-activating factor I, polypeptide chain, (2S)-2-amino-5-(carbamimidamido)pentanoic acid, ECTOL1, DPend, L-Methionine, Glycine Hydrochloride (2:1), Pedameth, ANTC, Search Engines, antp, Glycine Hydrochloride, iones, ipp2a2, imp alpha2, Acetylations, alpha2A-Kap, OCTD, Table, Salt Glycine, Glycin, 10^[-6], Imp-alpha2, taf-ibeta, grupos, great, site, Leimzucker, 2-aminoacetic acid, Long-Term Effect, AntP, ANTP, Controlled, Applications Software, Monoammonium Salt, Open Source, DYRK1, Controlling, data, Glycine Phosphate (1:1), Computer Software, DL-Methionine, protein complex, igaad, Aminoacetic acid, total expressed protein, Striated muscle RING zinc finger protein, DL Arginine Acetate, Peptide, group, Tripartite motif-containing protein 63, Software Tool, Glycine Carbonate (1:1), natural protein, Mus, Protein, I2PP2A, Hydrochloride, L Arginine, Dyrk1, l(3)84Ba, Software, Data Base, WMS2, Aminoacetic Acid, INSDC_feature:misc_binding, CC1, Engineering, Rest, Monolithium Salt, Protein Gene Products, Muscle-specific RING finger protein 1, dSET/TAF-Ibeta, 2610030F17Rik, Ion, Data, SSKS, alpha-amino-gamma-methylmercaptobutyric acid, Peptid, assay, variable, AA407739, 6-diaminohexanoic acid, groupemulticellular organismal catabolic process, single-organism catabolic process, SMRZ, SNDI, TFB1, Irreversible Electroporation, muscle system, Component of gems 4, Muscle hypotrophy, Dmp62, Ter94, protein, AI790512, sci, ALS14, d60, ter, OSIL, DmelCG2331, especially in the lower limbs, protein polypeptide chains, ref2p, IMP2, Roles, C130039E17Rik, Taf-6, HOW, How, Concepts, eIF-4-gamma 2, protein aggregate, DmTER94, IRF, l(3)j5D5, DmelCG6251, S2, 24B, Muscle Tissues, Ubiquitylation, RPN1, TERA, Rpn1, High Mobility Protein 20, Man (Taxonomy), reference sample, I, DmelCG32211, catabolism, Electric Field Mediated Cell Permeabilization, 15S Mg(2+)-ATPase p97 subunit, dvcp, Tissue, stru, Ubiquitin-related 1, proteins, VICKZ2, l(3)S053606, CG7762, p97/VCP, TfBl, CG10293, Irreversible, Natm1, TRAP2, Ligase, BTF2, APF-1, l(3)j5B5, IBMPFD, Amyotrophy, l(2)03775, 22.26, muscle wasting, Electric Field-Mediated Cell Permeabilization, ter94, Role Concepts, dVCP, 26S proteasome, Translation repressor NAT1, ubiquitination, STAP, IMP-2, RF1, eIF4G 2, Sam68, D7Ertd649e, TAF60/62, taf60, 0904/17, Yeti, Novel APOBEC-1 target 1, Iris RING finger protein, OSF-6, set of muscles, DmelCG10360, 6.3.2.-, rodents, Modern, ref, Neurogenic muscle atrophy, set of skeletal muscles, electropermeabilization, Taf[[II]]60, CG2331, Muscle RING finger protein 1, CEP52, AA589388, TAFII60, 3.6.4.6, dmTERA, Ref2P, p62B, ref(2)Po2, TAFII62, Tera, Role Concept, E130105L11Rik, SZ1, neurogenic, 1110001K06Rik, Role, BcDNA:GM02885, C77871, Sarcomere, Ubiq, Valosin-containing protein, Ubiquitin, proteasome, breakdown, l(3)76BDf, HEL-220, Human Ubiquitin, anon-WO2004063362.65, DYN4, TAF[[II]]60/62, TAF[[II]], anon-WO2004063362.67, muscle element, organ system, Reversible, Muscle atrophy, Osi, DmelCG7762, VCP/p97, CG6251, p97|VCP, Muscular atrophy, Apple, anon-EST:Liang-2.39, AAG1, TER ATPase, A170, humans, HC56, tibialis anterior muscle, PDB3, dTAF[[II]]60, HEL-S-70, skeletal muscle system., nat1, Reversible Electroporation, Peptidomics, Ref(2)P, P62, musculature system, HCAP1, muscles, eIF-4G 2, Gene, Synthetases, ATP Dependent Proteolysis Factor 1, IBSN, rodent, Ubiquitin carboxyl extension protein 80, protein-containing complex, body system, skeletal muscle, 62kDa, 4930547K17Rik, dTAF[[II]]62, Human, tibilais cranialis, HHRF-1, 7.12, BcDNA:LD21723, dTAF[[II]]262, Homo sapiens, polypeptide chain, p60, Ref(2)p, dap5, p62, dmTAF6, Gene Products, HMG-20, system, p68, ubiquitin-like protein modifier, TAFII60(62), Taf62, VCP ATPase, Man, DmelCG8151, DAP5, Taf60, Electropermeabilisation, study, VCP, Vcp, anatomical systems, anterior tibialis, AU045898, l(3)s2612, Tissues, TAF60, AW743425, STONE14, BTF2 p62, TAF[[II]]60, MuRF-1, Gemin-4, TAF[[II]]62, CG32211, CENP-29, 40S ribosomal protein S27a, Muscle degeneration, dTERA, musculus, CP27, ubiquitin, DmelCG10293, P97, ibialis anticus, dTAF62, Nat1, NAT1, ZIP3, TAF, protein tagging activity, SR3-4B, statistical analysis, musculus tibialis anterior, Controlled, ref(2)Pn, EIF4G2, striated muscle, l(2)06950, tibialis cranialis, Controlling, cdc48, DAP-5, degradation, musculi, taf6, protein complex, clone 2.39, p97, Proteins, SWC5, ref(2)p, Striated muscle RING zinc finger protein, qkr, dTAFII62, BUCENTAUR, Muscle, AW822074, l(3)S090417, Amyotrophy involving the extremities, l(2)06949, IBMPFD1, dme-TAFII60, Concept, RING finger protein 28, Tripartite motif-containing protein 63, somatic muscle, Ubiquitin A-52 residue ribosomal protein fusion product 1, TFIID 62, native protein, natural protein, Ubiquitin-related 2, KH93F, Protein, AI463667, MURF1, Synthetase, MURF2, TFIID, pTER94, TFIIH, ATP-Dependent Proteolysis Factor 1, connected anatomical system, BcDNA.GM02885, AA589433, who, covalent modifier, Muscle wasting, MuRF1, Murf1, muscles set, Neurogenic muscular atrophy, AI426861, DmelCG12042, Nupc1, CG9348, 3110001E05, CDC48, muscle, Who/How, Murf, BCNT, l(2)46Ch, Protein Gene Products, Muscle-specific RING finger protein 1, Gene Proteins, Rnf28, 60S ribosomal protein L40, Electroporation, dTAF6, l(2)46CFs, protein tag, ligase, Modern Man, CG8151, qkr[93F], musculature, Ubiquitin-related, regulation, RNF28, CG10360, Muscle Tissue, TAF6, l(2)46CFf, muscle group, skeletal muscle system, D130058I17RikNerve-Muscle Preparations, multicellular organismal catabolic process, single-organism catabolic process, SMRZ, SNDI, TFB1, Materials, Irreversible Electroporation, Laboratory, Component of gems 4, adult stage, Mus domesticus, Muscle hypotrophy, Dmp62, Ter94, CASP-14, protein, AI790512, sci, House Mouse, ALS14, d60, ter, OSIL, DmelCG2331, especially in the lower limbs, protein polypeptide chains, ref2p, IMP2, exact), Roles, C130039E17Rik, Neuromuscular Junctions, Taf-6, HOW, How, Concepts, eIF-4-gamma 2, protein aggregate, DmTER94, IRF, adult, l(3)j5D5, Myoneural Junction, DmelCG6251, S2, 24B, Nerve Muscle Preparation, Ubiquitylation, RPN1, TERA, Rpn1, High Mobility Protein 20, Man (Taxonomy), enzymes, I, DmelCG32211, catabolism, Electric Field Mediated Cell Permeabilization, 15S Mg(2+)-ATPase p97 subunit, dvcp, stru, Ubiquitin-related 1, Swiss Mice, proteins, VICKZ2, l(3)S053606, CG7762, p97/VCP, TfBl, CG10293, Irreversible, Natm1, TRAP2, Ligase, BTF2, APF-1, l(3)j5B5, IBMPFD, Amyotrophy, l(2)03775, NMJ, 22.26, muscle wasting, Electric Field-Mediated Cell Permeabilization, ter94, Role Concepts, dVCP, 26S proteasome, Translation repressor NAT1, ubiquitination, STAP, associated, IMP-2, RF1, eIF4G 2, Sam68, D7Ertd649e, TAF60/62, taf60, 0904/17, motor endplate, Yeti, Novel APOBEC-1 target 1, Iris RING finger protein, OSF-6, DmelCG10360, 6.3.2.-, rodents, Modern, mouse, ref, Neurogenic muscle atrophy, electropermeabilization, Taf[[II]]60, CG2331, Muscle RING finger protein 1, CEP52, AA589388, TAFII60, 3.6.4.6, dmTERA, Ref2P, p62B, ref(2)Po2, TAFII62, Tera, Role Concept, E130105L11Rik, stamen filament, SZ1, neurogenic, 1110001K06Rik, Mini-ICE, Role, Genetic Materials, BcDNA:GM02885, Adults, C77871, Genetic Material, Sarcomere, Ubiq, Valosin-containing protein, Ubiquitin, proteasome, Mus musculus, adults, breakdown, Caspase-14 subunit p10, mice, Swiss Mouse, l(3)76BDf, HEL-220, Human Ubiquitin, Caspase-14 subunit p19, anon-WO2004063362.65, MICE, Junction, DYN4, TAF[[II]]60/62, TAF[[II]], anon-WO2004063362.67, muscle element, domesticus, organ system, Contractile Protein, Reversible, filamento (Spanish, Muscle atrophy, Osi, DmelCG7762, VCP/p97, Material, CG6251, p97|VCP, Muscular atrophy, Biocatalyst, Myoneural, Apple, Cistron, anon-EST:Liang-2.39, Mouse, AAG1, TER ATPase, A170, humans, HC56, PDB3, dTAF[[II]]60, 3.4.22.-, HEL-S-70, skeletal muscle system., nat1, Reversible Electroporation, Peptidomics, Biocatalysts, Ref(2)P, P62, HCAP1, eIF-4G 2, Gene, Synthetases, mini-ICE, ATP Dependent Proteolysis Factor 1, IBSN, rodent, Ubiquitin carboxyl extension protein 80, protein-containing complex, body system, skeletal muscle, 62kDa, 4930547K17Rik, dTAF[[II]]62, Human, HHRF-1, 7.12, BcDNA:LD21723, dTAF[[II]]262, Homo sapiens, polypeptide chain, House, p60, Ref(2)p, dap5, p62, dmTAF6, Gene Products, Mus musculus domesticus, HMG-20, system, p68, ubiquitin-like protein modifier, TAFII60(62), Taf62, VCP ATPase, Mice, Man, DmelCG8151, DAP5, Taf60, Electropermeabilisation, study, VCP, Vcp, anatomical systems, Zea filament (narrow), AU045898, Genetic, l(3)s2612, Swiss, 花糸 (Japanese, TAF60, AW743425, STONE14, BTF2 p62, TAF[[II]]60, MuRF-1, Junctions, Gemin-4, TAF[[II]]62, CG32211, CENP-29, 40S ribosomal protein S27a, Muscle degeneration, Enzyme, Myoneural Junctions, dTERA, musculus, CP27, ubiquitin, DmelCG10293, P97, dTAF62, Nat1, NAT1, Preparation, ZIP3, TAF, protein tagging activity, SR3-4B, ref(2)Pn, EIF4G2, striated muscle, l(2)06950, Poaceae filament (narrow), cdc48, data, DAP-5, degradation, taf6, regulation of metabolism, protein complex, clone 2.39, regulation of organismal metabolic process, p97, regulation of multicellular organismal metabolic process, Proteins, SWC5, ref(2)p, enzyme activity, Striated muscle RING zinc finger protein, qkr, dTAFII62, BUCENTAUR, AW822074, Cistrons, l(3)S090417, Amyotrophy involving the extremities, l(2)06949, IBMPFD1, dme-TAFII60, Concept, Nerve-Muscle, RING finger protein 28, Tripartite motif-containing protein 63, somatic muscle, Ubiquitin A-52 residue ribosomal protein fusion product 1, TFIID 62, native protein, natural protein, Mus, Ubiquitin-related 2, KH93F, Protein, AI463667, MURF1, Synthetase, MURF2, TFIID, pTER94, TFIIH, ATP-Dependent Proteolysis Factor 1, connected anatomical system, BcDNA.GM02885, AA589433, who, covalent modifier, Muscle wasting, MuRF1, Murf1, Neurogenic muscular atrophy, AI426861, DmelCG12042, Neuromuscular, House Mice, Nupc1, CG9348, 3110001E05, CDC48, muscle, Who/How, Murf, BCNT, l(2)46Ch, Laboratory Mice, Protein Gene Products, Muscle-specific RING finger protein 1, Gene Proteins, Preparations, Rnf28, Nerve-Muscle Preparation, Contractile, 60S ribosomal protein L40, Electroporation, dTAF6, l(2)46CFs, protein tag, ligase, Modern Man, CG8151, qkr[93F], Ubiquitin-related, RNF28, CG10360, TAF6, Laboratory Mouse, l(2)46CFf, skeletal muscle system, D130058I17RikPeptidomics., SMRZ, MuRF1, Murf1, Iris RING finger protein, 6.3.2.-, number, Striated muscle RING zinc finger protein, Murf, Muscle RING finger protein 1, MuRF-1, skeletal muscle, presence, Muscle-specific RING finger protein 1, RING finger protein 28, Tripartite motif-containing protein 63, somatic muscle, count in organism, Rnf28, MURF1, MURF2, quantitative, RNF28, RF1, IRF, striated muscle, skeletal muscle system, presence or absence in organismfalseIdentification of the MuRF1 skeletal muscle ubiquitylome through quantitative proteomicsMuRF1 is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine if MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Tibialis anterior (TA) muscles were transfected with an untagged MuRF1 plasmid or control plasmid for 14 days. A total of 963 ubiquitination sites, corresponding to 250 proteins, were quantified from the TA muscle. Statistical analysis revealed that the overexpression of MuRF1 resulted in significant upregulation of 153 ubiquitination sites on 45 proteins and significant downregulation of 16 sites on 11 proteins. Substrates of MuRF1 include contractile and metabolic proteins, deubiquitinases, p62, and VCP. Moreover, MuRF1-mediated ubiquitination leads to destabilization and breakdown of the sarcomere and reveals a role for MuRF1 in the regulation of additional proteolytic pathways in skeletal 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