panoramaProteomicsPeder LundHomo SapiensMus Musculushttps://panoramaweb.org/MHqZI1.urlpxl446@case.eduCase Western Reserve UniversityPanoramaPublicThe eukaryotic genome is packaged around histone proteins, which are subject to a myriad of post-translational modifications. By controlling DNA accessibility and the recruitment of protein complexes that mediate chromatin-related processes, these modifications constitute a key mechanism of epigenetic regulation. Since mass spectrometry can easily distinguish between these different modifications, it has become an essential technique in deciphering the histone code. Although robust LC-MS/MS methods are available to analyze modifications on the histone N-terminal tails, routine methods for characterizing ubiquitin marks on histone C-terminal regions, especially H2AK119ub, are less robust. Here, we report the development of a simple workflow for the detection and improved quantification of the canonical histone ubiquitination marks H2AK119ub and H2BK120ub. The method entails a fully tryptic digestion of acid-extracted histones, followed by derivatization with heavy or light propionic anhydride. A pooled sample is then spiked into oppositely labeled single samples as a reference channel for relative quantification, and data is acquired using PRM-based nano-LC-MS/MS. We validated our approach with synthetic peptides as well as treatments known to modulate the levels of H2AK119ub and H2BK120ub. This new method complements existing histone workflows, largely focused on the lysine-rich N-terminal regions, by extending modification analysis to other sequence contexts.Optimized and Robust Workflow for Quantifying the Canonical Histone Ubiquitination Marks H2AK119ub and H2BK120ub by LC-MS/MS.Lopes Mariana M, Lund Peder J PJ, Garcia Benjamin A BAliquid chromatography tandem mass spectroscopy, Post Translational Amino Acid Modification, FBgn0003149, artificial sequence, single-organism developmental process, Lysine Hydrochloride, determination, postnatal development, growth and development, Visible Light, protein, Progress Reports, Protein Processing, Social Controls, Histone H2b, Polypeptides, Histone H2a, Techniques, Para, Lysine Acetate, peptido, Method, Summary Report, Code, CG9063, 2, Analysis, protein aggregate, Work Flow, CG5939, Formal Social Controls, Summary Reports, Chromatins, IKKg, Histone Mark, KEY, Key, L Lysine, Mass Spectrum Analysis, Post-Translational Protein, thymus nucleic acid, LCMSMS, High Mobility Protein 20, reference sample, peptides, Genomes, Analyses, Progress Report, Post-Translational, K, epsilon-diaminocaproic acid, Ubiquitin-related 1, procedures, Posttranslational Protein Processing, BcDNA:GH03694, Epigenomic, Social, APF-1, Progress, TNFSF14, Field Reports, LYS, Methodological Studies, Lysin, sample, chromosome scaffold, Histone H5, Therapies, Histone H4, Histone H7, Double-Stranded DNA, deoxyribonucleic acids, DNAn, SIMPLE, Histone H1, DmelCG9063, Histone H3, nuclear chromatin, LC-MS2, Therapy, Post-Translational Modification, anatomical protrusion, Radiation, UNQ391/PRO726, DmelCG5939, Modifications, acid, LC-MS/MS, Light, Double-Stranded, Procedure, CEP52, Spectrum Analysis, not genetically inherited, (Deoxyribonucleotide)n+m, Spectroscopy, DmIKKgamma, Workflows, Post Translational Modifications, LIGHT, Investigative Report, propanoic anhydride, prm, dIKK, Histone Modifications, PIG7, Kenny, lysine, l(3)S010605, desoxyribose nucleic acid, Ubiq, Ubiquitin, Visible Radiations, Acid, Visible Radiation, HVEML, Mark, Epigenetic, 0106/05, MS/MS, acide, Tails, BcDNAGH03694, Processing, PM/mPM, IKK-gamma, Field, Control, Spectrometry, Human Ubiquitin, acids, whole genome, Methodological, acido, alpha, Controls, Methodological Study, Treatments, 3L6, SYNTHETIC CONSTRUCT sequences, Report, DmelCG16910, Amino Acid Modification, Histone Marks, rich, spine, liquid chromatography-tandem mass spectroscopy, ds DNA, artificial, Posttranslational Modifications, Polypeptide, DNA, Epigenetics, Regulation, Posttranslational Modification, Work Flows, Regulations, cytoplasmic chromatin, Post-Translational Protein Modifications, DNS, Procedures, (Deoxyribonucleotide)n, Gene, ATP Dependent Proteolysis Factor 1, Spectrum Analyses, Ubiquitin carboxyl extension protein 80, protein-containing complex, dIKK-gamma, Deoxyribonucleic acids, LC-MS-MS, Posttranslational, protrusion, Human, Ly113, method, Investigative, Deoxyribonucleic Acid, mPM, DmIKK-gamma, method used in an experiment, LC-MSMS, Gene Products, Post Translational Modification, Mass, Studies, HMG-20, Post-Translational Modifications, Lysine, dmIKKgamma, synthetic genetic interaction (sensu inequality), IKK[[gamma]], Technique, Histone H3.3, Post Translational, Mass Spectroscopy, Modification, Marks, MS2, Double Stranded, synthetic genetic interaction defined by inequality, Deoxyribonucleic acid, Visible, Protein Modifications, Post-Translational Amino Acid Modification, Study, 40S ribosomal protein S27a, IKK, Posttranslational Amino Acid Modification, ubiquitin, Posttranslational Protein, peptidos, (Deoxyribonucleotide)m, TR2, Investigative Reports, protein tagging activity, Saeure, Controlled, TP53I7, Controlling, Formal Social Control, protein complex, DNAn+1, Proteins, Post-Translational Protein Modification, artificial gene, synthetic DNA, CD258, Histone, Peptide, development, IKKgamma, LC/MS/MS, MS, Ubiquitin A-52 residue ribosomal protein fusion product 1, native protein, Social Control, Ubiquitin-related 2, Acetate, Protein, Research Reports, chemical analysis, synthetic, sequence, techniques, ds-DNA, ATP-Dependent Proteolysis Factor 1, Protein Modification, L-Lysine, Mass Spectrum Analyses, tandem MS, Radiations, Mass Spectrum, covalent modifier, Dmikkgamma, Saeuren, HVEM-L, Post Translational Protein Processing, postnatal growth, Photoradiation, CG16910, synthetic constructs, Histone H1(s), sample population, LTg, Protein Gene Products, plan specification, Gene Proteins, Post Translational Protein Modification, Photoradiations, l(3)10631, 60S ribosomal protein L40, Reports, Therapeutic, Post-Translational Protein Processing, Desoxyribonukleinsaeure, liquid chromatography tandem mass spectrometry, primary structure of sequence macromolecule., Ubiquitin-related, Peptid, Treatment, regulation, assay, Summary, 6-diaminohexanoic acid, growth, Enisyl, Field Report, pm, PM, methodologyliquid chromatography tandem mass spectroscopy, LC-MS2, LC-MS/MS, liquid chromatography tandem mass spectrometry, LC-MS-MS., Workflows, LC/MS/MS, LCMSMS, Work Flow, liquid chromatography-tandem mass spectroscopy, Work Flows, LC-MSMSliquid chromatography tandem mass spectroscopy, Post Translational Amino Acid Modification, FBgn0003149, artificial sequence, single-organism developmental process, Lysine Hydrochloride, determination, postnatal development, growth and development, Visible Light, protein, Progress Reports, Protein Processing, Social Controls, Histone H2b, Polypeptides, Histone H2a, Techniques, Para, Lysine Acetate, peptido, Method, Summary Report, Code, CG9063, 2, Analysis, protein aggregate, Work Flow, CG5939, Formal Social Controls, Summary Reports, Chromatins, IKKg, Histone Mark, KEY, Key, L Lysine, Mass Spectrum Analysis, Post-Translational Protein, thymus nucleic acid, LCMSMS, High Mobility Protein 20, reference sample, peptides, Genomes, Analyses, Progress Report, Post-Translational, K, epsilon-diaminocaproic acid, Ubiquitin-related 1, procedures, Posttranslational Protein Processing, BcDNA:GH03694, Epigenomic, Social, APF-1, Progress, TNFSF14, Field Reports, LYS, Methodological Studies, Lysin, sample, chromosome scaffold, Histone H5, Therapies, Histone H4, Histone H7, Double-Stranded DNA, deoxyribonucleic acids, DNAn, SIMPLE, Histone H1, DmelCG9063, Histone H3, nuclear chromatin, LC-MS2, Therapy, Post-Translational Modification, anatomical protrusion, Radiation, UNQ391/PRO726, DmelCG5939, Modifications, acid, LC-MS/MS, Light, Double-Stranded, Procedure, CEP52, Spectrum Analysis, not genetically inherited, (Deoxyribonucleotide)n+m, Spectroscopy, DmIKKgamma, Workflows, Post Translational Modifications, LIGHT, Investigative Report, propanoic anhydride, prm, dIKK, Histone Modifications, PIG7, Kenny, lysine, l(3)S010605, desoxyribose nucleic acid, Ubiq, Ubiquitin, Visible Radiations, Acid, Visible Radiation, HVEML, Mark, Epigenetic, 0106/05, acide, Tails, BcDNAGH03694, Processing, PM/mPM, IKK-gamma, Field, Control, Spectrometry, Human Ubiquitin, acids, whole genome, Methodological, acido, alpha, Controls, Methodological Study, Treatments, 3L6, SYNTHETIC CONSTRUCT sequences, Report, DmelCG16910, Amino Acid Modification, Histone Marks, rich, spine, liquid chromatography-tandem mass spectroscopy, ds DNA, artificial, Posttranslational Modifications, Polypeptide, DNA, Epigenetics, Regulation, Posttranslational Modification, Work Flows, Regulations, cytoplasmic chromatin, Post-Translational Protein Modifications, DNS, Procedures, (Deoxyribonucleotide)n, Gene, ATP Dependent Proteolysis Factor 1, Spectrum Analyses, Ubiquitin carboxyl extension protein 80, protein-containing complex, dIKK-gamma, Deoxyribonucleic acids, LC-MS-MS, Posttranslational, protrusion, Human, Ly113, method, Investigative, Deoxyribonucleic Acid, mPM, DmIKK-gamma, method used in an experiment, LC-MSMS, Gene Products, Post Translational Modification, Mass, Studies, HMG-20, Post-Translational Modifications, Lysine, dmIKKgamma, synthetic genetic interaction (sensu inequality), IKK[[gamma]], Technique, Histone H3.3, Post Translational, Mass Spectroscopy, Modification, Marks, Double Stranded, synthetic genetic interaction defined by inequality, Deoxyribonucleic acid, Visible, Protein Modifications, Post-Translational Amino Acid Modification, Study, 40S ribosomal protein S27a, IKK, Posttranslational Amino Acid Modification, ubiquitin, Posttranslational Protein, peptidos, (Deoxyribonucleotide)m, TR2, Investigative Reports, protein tagging activity, Saeure, Controlled, TP53I7, Controlling, Formal Social Control, protein complex, DNAn+1, Proteins, Post-Translational Protein Modification, artificial gene, synthetic DNA, CD258, Histone, Peptide, development, IKKgamma, LC/MS/MS, MS, Ubiquitin A-52 residue ribosomal protein fusion product 1, native protein, Social Control, Ubiquitin-related 2, Acetate, Protein, Research Reports, chemical analysis, synthetic, sequence, techniques, ds-DNA, ATP-Dependent Proteolysis Factor 1, Protein Modification, L-Lysine, Mass Spectrum Analyses, Radiations, Mass Spectrum, covalent modifier, Dmikkgamma, Saeuren, HVEM-L, Post Translational Protein Processing, postnatal growth, Photoradiation, CG16910, synthetic constructs, Histone H1(s), sample population, LTg, Protein Gene Products, plan specification, Gene Proteins, Post Translational Protein Modification, Photoradiations, l(3)10631, 60S ribosomal protein L40, Reports, Therapeutic, Post-Translational Protein Processing, Desoxyribonukleinsaeure, liquid chromatography tandem mass spectrometry, primary structure of sequence macromolecule., Ubiquitin-related, Peptid, Treatment, regulation, assay, Summary, 6-diaminohexanoic acid, growth, Enisyl, Field Report, pm, PM, methodologyliquid chromatography tandem mass spectroscopy, LC-MS2, LC-MS/MS, liquid chromatography tandem mass spectrometry, LC-MS-MS., Workflows, LC/MS/MS, LCMSMS, Work Flow, liquid chromatography-tandem mass spectroscopy, Work Flows, LC-MSMSfalseAn optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MSThe eukaryotic genome is packaged around histone proteins, which are subject to a myriad of post-translational modifications. By controlling DNA accessibility and the recruitment of protein complexes that mediate chromatin-related processes, these modifications constitute a key mechanism of epigenetic regulation. Since mass spectrometry can easily distinguish between these different modifications, it has become an essential technique in deciphering the histone code. Although robust LC-MS/MS methods are available to analyze modifications on the histone N-terminal tails, routine methods for characterizing ubiquitin marks on histone C-terminal regions, especially H2AK119ub, are less robust. Here we report the development of a simple workflow for the detection and improved quantification of the canonical histone ubiquitination marks H2AK119ub and H2BK120ub. The method entails a fully tryptic digestion of acid-extracted histones followed by derivatization with heavy or light propionic anhydride. A pooled sample is then spiked into oppositely labeled single samples as a reference channel for relative quantification, and data is acquired using PRM-based nanoLC-MS/MS. We validated our approach with synthetic peptides as well as treatments known to modulate the levels of H2AK119ub and H2BK120ub. This new method complements existing histone workflows, largely focused on the lysine-rich N-terminal regions, by extending modification analysis to other sequence contexts.Tue Dec 10 00:00:00 GMT 2024PXD05321210090960639556659