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Proteomic analysis of assembled chromatin with SWATH-MS
Proteomic analysis of assembled chromatin with SWATH-MS
In all eukaryotes, the large DNA molecule must be packed into the nucleus of a cell in a very compact manner. The compacted structure consisting of histones, non-histone proteins and nucleic acids is called chromatin. To transcribe, repair or replicate the DNA, chromatin must be regulated in a highly dynamic manner. The regulators are mostly proteins that can remodel and assemble chromatin by altering the accessibility for DNA-templated processes. Acquired results in this thesis show temporal changes in protein binding using an in vitro reconstitution system based on Drosophila melanogaster embryos for 15 min, 1 h and 4 hrs of chromatin assembly. In addition, newly synthesized DNA and its associated proteins were isolated after replication by nascent chromatin capture (NCC) in human cell lines and quantified at various times of chromatin assembly. Results from a comparison of both systems indicate that the principles of chromatin assembly are conserved between Homo sapiens and Drosophila melanogaster. In addition, results from both approaches show the association of proteasomal proteins with chromatin that are well-known for their function in protein degradation. Proteasome inhibition causes protein aggregates to form during assembly indicating an important role of the proteasome during chromatin assembly. Both experimental methods rely on the analysis of proteins with mass spectrometry. Comparative experiments using different mass spectrometric techniques show that using a data-independent acquisition method (SWATH-MS) greatly improves the number of identified and quantified proteins in comparison to classical data-dependent techniques, thereby facilitating the downstream statistical analysis. Data presented here, contribute new insights about the binding kinetics of proteins during chromatin assembly and suggest that the proteasome functions as a quality control during chromatin assembly.
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Völker-Albert, Moritz
2017
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Völker-Albert, Moritz (2017): Proteomic analysis of assembled chromatin with SWATH-MS. Dissertation, LMU München: Faculty of Medicine
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Abstract

In all eukaryotes, the large DNA molecule must be packed into the nucleus of a cell in a very compact manner. The compacted structure consisting of histones, non-histone proteins and nucleic acids is called chromatin. To transcribe, repair or replicate the DNA, chromatin must be regulated in a highly dynamic manner. The regulators are mostly proteins that can remodel and assemble chromatin by altering the accessibility for DNA-templated processes. Acquired results in this thesis show temporal changes in protein binding using an in vitro reconstitution system based on Drosophila melanogaster embryos for 15 min, 1 h and 4 hrs of chromatin assembly. In addition, newly synthesized DNA and its associated proteins were isolated after replication by nascent chromatin capture (NCC) in human cell lines and quantified at various times of chromatin assembly. Results from a comparison of both systems indicate that the principles of chromatin assembly are conserved between Homo sapiens and Drosophila melanogaster. In addition, results from both approaches show the association of proteasomal proteins with chromatin that are well-known for their function in protein degradation. Proteasome inhibition causes protein aggregates to form during assembly indicating an important role of the proteasome during chromatin assembly. Both experimental methods rely on the analysis of proteins with mass spectrometry. Comparative experiments using different mass spectrometric techniques show that using a data-independent acquisition method (SWATH-MS) greatly improves the number of identified and quantified proteins in comparison to classical data-dependent techniques, thereby facilitating the downstream statistical analysis. Data presented here, contribute new insights about the binding kinetics of proteins during chromatin assembly and suggest that the proteasome functions as a quality control during chromatin assembly.