MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/x01/MSV000079757/primaryOK2000000ProteomicsDr Geir SlupphaugLTQ Orbitrap EliteHomo Sapiens (ncbitaxon:9606)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=1c899a0b3b5543318412323d54773ca5MassIVE29MOD:00544 - "A protein modification that effectively converts a residue containing common isotopes to a 6x(13)C labeled residue."Transient transfection of mammalian cells with plasmid expression vectors and chemical transfection reagents is widely used to study protein transport and dynamics as well as phenotypic alterations mediated by the overexpressed protein. Despite the undisputed impact of this technique, surprisingly little is known about the cellular effects mediated by the transfection process per se. Conceivably, off-target effects could have implications upon proteins or processes being studied and understanding the molecular pathways affected would add value to the interpretation of experimental observations subsequent to cell transfection. Here we have used a SILAC-based proteomic approach to study differentially expressed proteins after transfection of HeLa cells with ECFP vector using a commonly employed non-liposome based transfection reagent, Fugene®HD. Whereas the transfection reagent itself mediated minimal effects upon protein expression, 11 proteins were found to be significantly upregulated after transfection, all of which were associated with an interferon type I/II response. The upregulated proteins might potentially inflict major cellular processes such as RNA splicing, chromatin remodeling, post-translational protein modification and cell cycle control. The results were validated by western analysis as well as quantitative RT-PCR and this demonstrated that an essentially identical response was induced in HeLa by transfection using an empty pUC18 vector, which does not contain a mammalian virus promoter, as well as a liposome-based transfection reagent, Lipofectamine(TM)2000. Notably, no induction of the interferon response was observed in HEK293 cells, suggesting that these cells might be preferable to HeLa to avoid undesired off-target effects in transfection studies encompassing interferon-signaling and antiviral responses.Off-target responses in the HeLa proteome subsequent to transient plasmid-mediated transfection.Hagen Lars L, Sharma Animesh A, Aas Per Arne PA, Slupphaug Geir GSquatter, Episomes, transfect, Migrant Worker, Transfections., Migrant, Nomads, Transients, HeLa, Squatters, Nomad, Plasmid, Nonmigrant, total expressed protein, Migrants, RCB0007, Nonmigrants, Workers, Episome, Worker, Proteomes, Transient, Migrants and Transients, Migrant Workersprojections, protein translation, Post Translational Amino Acid Modification, posttranslational modification, Post-Translational Protein Modifications, tumor suppressor, lamellae, Cell Cycle Control, Gene, Virus Infection, regulation of cell cycle transition, Progress Reports, Virus Disease, process of organ, Protein Processing, Arrests, Posttranslational, lamella, Cell Cycle-Transition Points, cell cycle regulator, Investigative, Summary Report, responsivity, Gene Products, Virus, Post Translational Modification, Virus Infections, Mood, Cell Cycle Transition Points, Post-Translational Modifications, Type I, Summary Reports, Post Translational, Modification, Post-Translational Protein, reactivity, viral infection, Interferons, posttranslational amino acid modification, Viral Diseases, Viruses, Progress Report, Infections, Post-Translational, Moods, Interferons Type I, ridges, posttranslational protein modification, Posttranslational Protein Processing, Protein Modifications, Post-Translational Amino Acid Modification, Progress, Field Reports, control of cell cycle progression, regulation of progression through cell cycle, regulation of cell cycle progression, Posttranslational Amino Acid Modification, papilla, Posttranslational Protein, Checkpoint, Cell Cycle, Cell Cycle-Transition, Investigative Reports, Cell Cycle Arrest, laminae, Viral Infection, cell cycle modulation, Post-Translational Modification, Disease, protein anabolism, Viral, protein biosynthetic process, Cell Cycle-Transition Point, Affects, Proteins, Modifications, lamina, flanges, Post-Translational Protein Modification, infections, Cell, Post Translational Modifications, Checkpoints, Investigative Report, Research Reports, Protein, Diseases, shelf, Infection, modulation of cell cycle progression, protein formation, protein biosynthesis, interferon type I, Cell Cycle Arrests, Protein Modification, flange, organ process, Arrest, IFN type I, PTM, Cell Cycle Controls, cell cycle control, Protein., Viral Infections, Post Translational Protein Processing, shelves, Viral Disease, Processing, Field, Control, Controls, projection, ridge, post-translational amino acid modification, Cell Cycle Checkpoint, Protein Gene Products, Gene Proteins, processes, Cycle-Transition Point, Report, Post Translational Protein Modification, Amino Acid Modification, Reports, protein synthesis, Type I Interferon, Point, post-translational modification, Type I Interferons, Post-Translational Protein Processing, virulence, Posttranslational Modifications, response, Summary, Interferon, cell cycle regulation, Posttranslational Modification, Field ReportSquatter, Episomes, transfect, Migrant Worker, Transfections., Migrant, Nomads, Transients, HeLa, Squatters, Nomad, Plasmid, Nonmigrant, total expressed protein, Migrants, RCB0007, Nonmigrants, Workers, Episome, Worker, Proteomes, Transient, Migrants and Transients, Migrant Workersprojections, anti-viral agents, antivirals, Post Translational Amino Acid Modification, Viridae, 293T Cell, LF 2000, Ghrfr, 293 Cells, posttranslational modification, AU023367, tumor suppressor, determination, FuGene 6, Human Embryonic Kidney Cell Line 293, regulation of cell cycle transition, RT-PCR, protein, Antiviral, Antiviral Agent, Protein Processing, Arrests, Chromatin Assembly, 1B1, dmTAF[[II]]230, anon-EST:Posey9, protein polypeptide chains, Readability, Cell Cycle-Transition Points, cell cycle regulator, responsivity, Virus, Cell Cycle Transition Points, HEK 293, protein aggregate, ATP-dependent chromatin remodeling, imprinted and ancient gene protein, Post-Translational Protein, Interferons, posttranslational amino acid modification, Gated Protein Transport, Protein Trafficking, Migrant Worker, TFIID TAF250, cel, Post-Translational, Interferons Type I, Nonmigrant, Chromatin Remodeling, proteins, Posttranslational Protein Processing, Transient, regulation of cell cycle progression, Vira, papilla, Transmembrane Protein Transport, Squatters, Cell Cycle, RCB0007, l(2)01103, Reagents, Cell Cycle Arrest, Transfections, HtsF, reagents, 293 Cell, single organism signaling, Add, ADD, Post-Translational Modification, dTAF[[II]]230, antiviral agents, anatomical protrusion, Modifications, lamina, TAF200, flanges, lit, Protein Targeting, anti-viral 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transport, experimental, lamellae, Reagent, Disassembly, Cell Cycle Control, Ovhts, Chromatin Disassemblies, number, Gene, HtsRC, dTAFII250, CG9325, HEK293 Cell, protein-containing complex, Ximpact, EfW1, process of organ, Xt, presence, Migrant Workers, 293T, protrusion, Posttranslational, Agent, lamella, polypeptide chain, dmTAF1, Add-hts, Taf230, Messenger, Gene Products, Post Translational Modification, l(2)k14523, l(2)00634, Post-Translational Modifications, Ovhts-RC, Animal, Type I, Chromatin Disassembly, Reagents and Indicators, Post Translational, TAF250, Drugs, Modification, study, reactivity, Taf200, Animal Virus, HTS-R1, methods, dTAF[[II]]250, reactivo, Viruses, cell, experimental section, Transients, Protein Translocation, HeLa, Nonmigrants, HTS-RC, Taf1p, ridges, posttranslational protein modification, Worker, Indicators, Protein Modifications, Squatter, Post-Translational Amino Acid Modification, dTAF250, control of cell cycle progression, regulation of progression through cell cycle, Posttranslational Amino Acid Modification, Protein Transport, LF-2000, 293T Cells, Posttranslational Protein, Checkpoint, Cell Cycle-Transition, TAF, laminae, CG34197, DmelCG43443, Hts-RC, cell cycle modulation, RNA, TAF[[II]]250, Cell Cycle-Transition Point, protein complex, Proteins, Post-Translational Protein Modification, Human Kidney Cell Line 293, l(3)84Ab, BG:DS00004.13, Antiviral Drugs, Traffickings, Cell, impact-a, Splicings, dTAF230, RNA Splicings, viruses, count in organism, Checkpoints, native protein, GLI3-190, HeLa Cell, natural protein, p230, Protein, chemical analysis, Nomad, TAF[[II]]250/230, IMPACT, imprinted and ancient gene protein homolog, TFIID, Dmel_CG34197, Protein Localization Processes, Protein Modification, Cell Cycle Arrests, Zoophaginae, Bph, flange, INSDC_feature:regulatory, organ process, Taf[[II]]250, Chromatin, pre-mRNA splicing factor activity, TAF[[II]]230, Cell Cycle Controls, cell cycle control, adducin, Chromatin Modeling, Post Translational Protein Processing, HEK293, lipofectamine 2000, Antiviral Drug., TAF[II]250, Episome, Understanding, post-translational amino acid modification, ATP-dependent chromatin remodelling, Cell Cycle Checkpoint, Protein Gene Products, Drug, Episomes, Gene Proteins, processes, Cellular Protein Targeting, Post Translational Protein Modification, Vesicular Protein Transport, DmelCG17603, signalling process, Point, Nomads, Post-Translational Protein Processing, Plasmid, Attention Deficit Hyperactivity Disorder, assay, response, reverse transcription polymerase chain reaction, EST D, RWDD5, TAF10PXD001278trueOff-target responses in the HeLa proteome subsequent to transient plasmid transfection using SILACWe report a significant differential expression of proteins, all of which are associated with a viral infection response typified by Interferon type I/II. Notably the upregulated proteins may affect several vital cellular processes, including cell cycle control, translation and post-translational modification of proteins.Mon May 23 12:46:00 BST 2016MSV00007975725448019