{"database":"MassIVE","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Other":["ftp://massive-ftp.ucsd.edu/x01/MSV000079550/"]},"type":"primary"},"statusCodeValue":200,"statusCode":"OK"}],"scores":{"citationCount":0,"reanalysisCount":0,"viewCount":0,"searchCount":0},"additional":{"omics_type":["Proteomics"],"submitter":["Dr Hendrik G. Stunnenberg"],"instrument_platform":["Q Exactive"],"species":["Homo Sapiens (ncbitaxon:9606)"],"full_dataset_link":["https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=fd62097ba5984b42a61efd75acd76ed1"],"sample_protocol":[""],"repository":["MassIVE"],"file_size":["93"],"ptm_modification":["MOD:00397 - \"A protein modification that is produced by reaction with iodoacetamide, usually replacement of a reactive hydrogen with a methylcarboxamido group.\""],"data_protocol":[""],"pubmed_abstract":["Interactions between transcription factors and genomic DNA, and in particular their impact on disease and cell fate, have been extensively studied on a global level using techniques based on next-generation sequencing. These approaches, however, do not allow an unbiased study of protein complexes that bind to certain DNA sequences. DNA pulldowns from crude lysates combined with quantitative mass spectrometry were recently introduced to close this gap. Established protocols, however, are restricted to cell lines because they are based on metabolic labeling or require large amounts of material. We introduce a high-throughput-compatible DNA pulldown that combines on-bead digestion with direct dimethyl labeling or label-free protein quantification. We demonstrate that our method can efficiently identify transcription factors binding to their consensus DNA motifs in extracts from primary foreskin fibroblasts and peripheral blood mononuclear cells (PBMCs) freshly isolated from human donors. Nuclear proteomes with absolute quantification of nearly 7000 proteins in K562 cells and PBMCs clearly link differential interactions to differences in protein abundance, hence stressing the importance of selecting relevant cell extracts for any interaction in question. As shown for rs6904029, a SNP highly associated with chronic lymphocytic leukemia, our approach can provide invaluable functional data, for example, through integration with GWAS."],"pubmed_title":["A quantitative proteomics tool to identify DNA-protein interactions in primary cells or blood."],"pubmed_authors":["Hubner Nina C NC, Nguyen Luan N LN, Hornig Nadine C NC, Stunnenberg Hendrik G HG"],"name_synonyms":["portion of blood, thymus nucleic acid, DNS, whole blood, (Deoxyribonucleotide)n, Peptidomics, vertebrate blood., protein complex, DNAn+1, Proteins, number, Double Stranded, Gene, Deoxyribonucleic acid, proteins, protein, Double-Stranded, protein-containing complex, presence, Cell, Deoxyribonucleic acids, Protein Gene Products, (Deoxyribonucleotide)n+m, Gene Proteins, count in organism, protein polypeptide chains, native protein, Deoxyribonucleic Acid, natural protein, polypeptide chain, ds DNA, Protein, Desoxyribonukleinsaeure, Gene Products, Double-Stranded DNA, (Deoxyribonucleotide)m, DNA, deoxyribonucleic acids, ds-DNA, DNAn, protein aggregate, desoxyribose nucleic acid"],"description_synonyms":["liquid chromatography tandem mass spectroscopy, Forms, scale tissue, rasGAP, Peripheral Blood Mononuclear Cells, Laboratory, 10538, Mbp1, protein, GAP1, temporal, prevention, protein polypeptide chains, Techniques, Background, GADPH, diseases, CG8893, gap1, Method, Cultural, Line, Extracts, PKWS, diseases and disorders, Peripheral Blood Mononuclear Cell, Analysis, protein aggregate, myd, prevention and control, FBgn 32821, imprinted and ancient gene protein, Mass Spectrum Analysis, ethnicity, human disease, thymus nucleic acid, LCMSMS, Gene Expressions, Man (Taxonomy), reference sample, Analyses, FATE, plant peltate hair, Gapd, Tissue, Mbp-1, mip, proteins, free, preventive measures, d-CdGAPr, Mononuclear, Methodological Studies, Mononuclear Leukocytes, CM-AVM, Homo sapiens disease, Double-Stranded DNA, Consensus Development, Tissue Donor, deoxyribonucleic acids, DNAn, DmelCG8893, LC-MS2, close to, preventive therapy, Transplant Donor, gyltl1b-b, Modern, Transplant, LC-MS/MS, Semen Donors, Double-Stranded, peltate hair., Procedure, PBMC Peripheral Blood Mononuclear Cells, RASA, Spectrum Analysis, (Deoxyribonucleotide)n+m, Spectroscopy, Cultural Background, Cultures, MDDGA6, mKIAA0609, sxt, Diseases, CT43, Donors, GAP, Gap, desoxyribose nucleic acid, p120RASGAP, KIAA0609, Lines, fg, Ras-GAP, CG10538, Factors, gyltl1b, Cultural Beliefs, mdc1d, Spectrometry, expanded, gap, Methodological, Methodological Study, human, Semen Donor, disease, MDC1D, Semen, Cell Extract, enr, enlarged, liquid chromatography-tandem mass spectroscopy, label, Ras p21 protein activator, ds DNA, Cells, prepuce of male, DNA, E430016J11Rik, Ovum, Ovum Donors, Organ Donors, Proteomes, Fibroblast, humans, big, other disease, human being, DNS, Procedures, (Deoxyribonucleotide)n, Extract, GAPDH II, penile prepuce, number, GTPase-activating protein, Gene, Gapdh13F, Spectrum Analyses, Development, protein-containing complex, Transcription Factor, Ximpact, presence, Deoxyribonucleic acids, froggy, gDNA, LC-MS-MS, Gyltl1a, Human, method, large, preputium penis, polypeptide chain, Deoxyribonucleic Acid, Homo sapiens, method used in an experiment, LC-MSMS, Consensus, Gene Products, Mass, Studies, disease or disorder, Technique, Man, Mass Spectroscopy, Backgrounds, study, Transcription, RasGAP, prepuce, MDDGB6, ligand, GA3PDH, Gap 1, Double Stranded, Cultural Relativisms, Deoxyribonucleic acid, labeling, LARGE, GAPDH2, Expressions, Transplant Donors, non-neoplastic, Study, BPFD#36, preputium, Gapdh-2, Mononuclear Leukocyte, great, Customs, disorder, CG6721, Expression, (Deoxyribonucleotide)m, culture, Ovum Donor, incidence, Controlled, Organ, Controlling, Foreskins, Leukocyte, bead, protein complex, DNAn+1, Proteins, disorders, Cell Lines, Factor, medical condition, Cultural Backgrounds, RASGAP, impact-a, Cell, near to, count in organism, K562, LC/MS/MS, MS, native protein, natural protein, Gapdh, prepucium, Protein, sequence, IMPACT, imprinted and ancient gene protein homolog, condition, Donor, rI533, ds-DNA, scales, p120GAP, Mass Spectrum Analyses, GAPDH, Mass Spectrum, Peripheral Blood Human Mononuclear Cells, DmelCG6721, scale, Organ Donor, prophylaxis, DmelCG10538, CMAVM, primary structure of sequence macromolecule, Protein Gene Products, plan specification, foreskin, Gene Proteins, control, approaches, Modern Man, cardinality, vicinity of, Desoxyribonukleinsaeure, praeputium, Relativisms, liquid chromatography tandem mass spectrometry, Relativism, Cultural Relativism, RWDD5"],"pubmed_title_synonyms":["portion of blood, thymus nucleic acid, DNS, whole blood, (Deoxyribonucleotide)n, Peptidomics, vertebrate blood., protein complex, DNAn+1, Proteins, Double Stranded, Gene, Deoxyribonucleic acid, proteins, protein, Double-Stranded, protein-containing complex, Cell, Deoxyribonucleic acids, Protein Gene Products, (Deoxyribonucleotide)n+m, Gene Proteins, protein polypeptide chains, native protein, Deoxyribonucleic Acid, natural protein, polypeptide chain, ds DNA, Protein, Desoxyribonukleinsaeure, Gene Products, Double-Stranded DNA, (Deoxyribonucleotide)m, DNA, deoxyribonucleic acids, ds-DNA, DNAn, protein aggregate, desoxyribose nucleic acid"],"pubmed_abstract_synonyms":["rasGAP, Nucleotide Sequencing, Peripheral Blood Mononuclear Cells, RNA Sequence, 10538, Chronic B-Lymphocytic, CG30327, GAP1, dmTAF[[II]]230, B Cell, Small, protein polypeptide chains, Method, gap1, Line, Extracts, Lymphoplasmacytoid Lymphoma, Analysis, Chronic Lymphocytic Leukemias, myd, FBgn 32821, Lymphomas, Deep Sequencing, imprinted and ancient gene protein, thymus nucleic acid, TFIID TAF250, Analyses, cel, FATE, Illumina Sequencing, Gapd, Tissue, CLL Lymphoplasmacytoid Lymphoma, Mbp-1, Small Lymphocytic Lymphoma, Well-Differentiated Lymphocytic Lymphoma, proteins, procedures, B Cell., Mononuclear, CM-AVM, Homo sapiens disease, Tissue Donor, Nucleotide, DmelCG8893, B-Cell Chronic Lymphocytic Leukemia, dTAF[[II]]230, Transplant Donor, Transplant, B Cell Malignancy, TAF200, Semen Donors, Low-Grade B-Cell Malignancies, Procedure, PBMC Peripheral Blood Mononuclear Cells, RASA, Spectrum Analysis, lymphoplasmacytic leukemia, mKIAA0609, Next-Generation, Donors, B Cell Chronic Lymphocytic Leukemia, chronic lymphatic leukemia, DNA Sequence, B Cell Leukemia, desoxyribose nucleic acid, fg, Ras-GAP, Dmel_CG6393, CG10538, Lymphatic Leukemias, Spectrometry, expanded, Methodological, Chronic Lymphoblastic Leukemias, human, Semen Donor, High Throughput Sequencing, MDC1D, Cell Extract, enr, Taf250, CLL Lymphoplasmacytoid, Lymphoma, ds DNA, Cells, DNA, E430016J11Rik, Organ Donors, TAF230, Fibroblast, humans, Nucleotide Sequence, big, CLL, DNS, (Deoxyribonucleotide)n, DNA Motifs, Extract, B-Lymphocytic Leukemias, chronic LYMPHOCYTIC, Gapdh13F, High-Throughput DNA, protein-containing complex, Transcription Factor, Next-Generation Sequencing, Human, large, preputium penis, Deoxyribonucleic Acid, Deep, Consensus, Gene Products, disease or disorder, Chronic B-Cell Leukemias, Well-Differentiated Lymphocytic, Technique, Man, Low-Grade, Transcription, Leukemia, dTAF[[II]]250, RasGAP, cell, MDDGB6, ligand, Gap 1, Double Stranded, Deoxyribonucleic acid, Pyrosequencing, GAPDH2, Sequencing, Transplant Donors, Diffuse, Study, dTAF250, Disrupted In B Cell Malignancy, Mononuclear Leukocyte, DNA Motif, 65K, CG42257, Dmel_CG30327, CG6721, Chronic, Diffuse Well Differentiated Lymphocytic Lymphoma, snp, (Deoxyribonucleotide)m, Chronic B-Lymphocytic Leukemias, Ovum Donor, Diffuse Well-Differentiated Lymphocytic Lymphoma, Organ, leukemia, RNA, cg11478, Foreskins, whole blood, DNAn+1, Small Cell, Proteins, disorders, Chronic Lymphocytic Leukemia, Chronic B-Lymphocytic Leukemia, lymphoplasmacytic leukaemia, Factor, BG:DS00004.13, Chronic Lymphoblastic Leukemia, Cell, Leukemias, chronic, dTAF230, Sequences, K562, MS, Well-Differentiated, Chronic Lymphatic, native protein, Small Cell Lymphoma, Gapdh, prepucium, Lymphocytic Leukemias, TAF[[II]]250/230, IMPACT, imprinted and ancient gene protein homolog, condition, Donor, rI533, p120GAP, Low-Grade B-Cell, Mass Spectrum Analyses, GAPDH, Next Generation Sequencing, RNA Sequences, Chronic Lymphatic Leukemia, Taf[[II]]250, Peripheral Blood Human Mononuclear Cells, DmelCG6721, Peripheral Blood, Lymphocytic Lymphomas, DmelCG10538, CMAVM, plan specification, Reticuloendothelial System, Gene Proteins, Desoxyribonukleinsaeure, High Throughput Nucleotide Sequencing, High-Throughput Nucleotide, CLL Lymphoplasmacytoid Lymphomas, Chronic B-Cell, Massively-Parallel, Blood, Mbp1, LFQ, Lymphatic Leukemia, protein, CG11478, Techniques, GADPH, High-Throughput RNA Sequencing, diseases, CG8893, PKWS, diseases and disorders, Peripheral Blood Mononuclear Cell, protein aggregate, chronic lymphatic, Mass Spectrum Analysis, DmelCG42257, human disease, Man (Taxonomy), B-cell chronic lymphocytic leukemia, Small-Cell Lymphomas, Plasmacytoid, mip, B-Lymphocytic Leukemia, Base Sequences, Lymphoblastic Leukemias, d-CdGAPr, Chronic Lymphatic Leukemias, Methodological Studies, High Throughput RNA Sequencing, Mononuclear Leukocytes, Nucleotide Motif, Double-Stranded DNA, Consensus Development, Malignancies, deoxyribonucleic acids, DNAn, chronic lymphatic leukaemia, High-Throughput RNA, gyltl1b-b, Modern, High-Throughput DNA Sequencing, Double-Stranded, TAFII-250, TAF250/230, (Deoxyribonucleotide)n+m, Spectroscopy, TAFII250, Ion Torrent Sequencing, MDDGA6, Massively-Parallel Sequencing, CG6393, sxt, Diseases, CT43, B-Cell Malignancies, B Lymphocytic Leukemia, RNA Motifs, Ion Torrent, GAP, Gap, lymphocytic, p120RASGAP, KIAA0609, Lines, Lymphoblastic Leukemia, Factors, gyltl1b, Well-Differentiated Lymphocytic Lymphomas, mdc1d, Disrupted In B-Cell Malignancy, Chronic Lymphoblastic, gap, CG17603, TAF[[II]], Methodological Study, Low-Grade B-Cell Malignancy, disease, Semen, enlarged, SR3-5, Lymphoplasmacytoid, Ras p21 protein activator, PBMCs, Base, chronic lymphocytic leukaemia, prepuce of male, Ovum, Ovum Donors, Proteomes, Small Lymphocytic Lymphomas, Lymphoplasmacytoid Lymphomas, other disease, d230, Chronic B-Cell Leukemia, High Throughput DNA Sequencing, human being, Procedures, Lymphocytic Leukemia, GAPDH II, penile prepuce, GTPase-activating protein, Gene, B-Cell, dTAFII250, Spectrum Analyses, Development, Ximpact, EfW1, Illumina, B-cell chronic lymphocytic leukaemia, Deoxyribonucleic acids, froggy, gDNA, Gyltl1a, method, polypeptide chain, Homo sapiens, dmTAF1, Taf230, B-cell chronic lymphoid leukemia, method used in an experiment, PBMC, Studies, Mass, Massively Parallel Sequencing, Well Differentiated, RNA Motif, Mass Spectroscopy, TAF250, Lymphocytic, study, Taf200, Ion Proton Sequencing, Malignancy, prepuce, GA3PDH, Taf1p, labeling, LARGE, Small-Cell Lymphoma, non-neoplastic, BPFD#36, preputium, Gapdh-2, great, disorder, High-Throughput, RNA Sequencing, Motifs, TAF, Chronic Lymphocytic, Low Grade, TAF[[II]]250, Leukocyte, bead, protein complex, Nucleotide Sequences, Cell Lines, B-Cell Malignancy, l(3)84Ab, medical condition, RASGAP, impact-a, B-Cell Leukemia, natural protein, Sequence, p230, DNA Sequencing, Protein, TFIID, techniques, ds-DNA, Mass Spectrum, small lymphocytic lymphoma, TAF[[II]]230, Organ Donor, B-Cell Leukemias, Lymphoblastic, TAF[II]250, Small Lymphocytic, Protein Gene Products, foreskin, Small-Cell, DmelCG17603, Lymphocytic Lymphoma, High-Throughput Sequencing, Modern Man, praeputium, DNA Sequences, Ion Proton, RWDD5, methodology, Motif, TAF1"],"citation_count":["0"],"additional_accession":["PXD000782"]},"is_claimable":true,"name":"A quantitative proteomics tool to identify DNA-protein interactions in primary cells or blood","description":"The interaction between transcription factors and genomic DNA forms the basis for spatio-temporal control of gene expression. Therefore, these interactions and their impact on disease and cellular fate are extensively studied on a global level, mainly using techniques based on next-generation sequencing. These techniques, however, do not allow an unbiased study of proteins or entire protein complexes that bind to a certain DNA sequence. In recent years, DNA pull-downs followed by quantitative mass spectrometry were introduced to close this gap. Established protocols, however, are based on metabolic labeling techniques or require enormous amounts of cellular material, thus restricting the method to cell lines grown in culture. Furthermore, they require substantial amount of expertise, thus keeping this technique restricted to a limited number of laboratories. Here, we introduce a high-throughput compatible, LC-MS/MS based DNA pull-down that combines on-bead digestion with direct dimethyl labeling or label-free protein quantification. We demonstrate, that our method can efficiently identify transcription factors binding to their known consensus DNA motifs when using nuclear extracts from model cell lines. Subsequently, we apply the method to study DNA-protein interactions in primary foreskin fibroblasts and peripheral blood mononuclear cells (PBMCs) freshly isolated from human donors. We show that the same DNA sequence binds different sets of proteins in an established model cell line as opposed to PBMCs. This stresses the importance of selecting relevant cell extracts for any interaction in question. In-depth nuclear proteomes with absolute quantification of close to 7,000 proteins in K562 cells and PBMCs clearly link these differential interactions to differences in protein abundance. In conclusion, our approach, applicable to primary material and capable of profiling DNA-protein interactions in high-throughput, will likely prove itself as a useful screening platform and will provide invaluable functional data, for example through integration with large scale GWAS.","dates":{"publication":"Tue Mar 01 13:46:00 GMT 2016"},"accession":"MSV000079550","cross_references":{"pubmed":["25546135"]}}