NOV 23, 2016 8:00 AM PST

Studying Kinetics of Chromatin Assembly with SWATH-MS

Sponsored by: SCIEX OMICS, SCIEX OMICS
Speakers
  • PhD candidate working on the Kinetics of Chromatin Assembly combined with Mass spectrometry
    Biography
      As PhD student in the quantitative proteomics laboratory of Prof. Axel Imhof at the LMU in Munich, Moritz Völker-Albert investigates the kinetics of chromatin assembly. Therefore, he applies data-dependent as well as data-independent methods to describe time-dependent proteomic changes during chromatin maturation by means of an in-vitro assembly protocol.
      Prior to starting his PhD in Munich in 2013, Moritz Völker-Albert successfully finished his studies in Biomedical Sciences at the Universities of Leiden and Utrecht in the Netherlands with a master degree and from the University of Marburg in Germany with a Bachelor degree. During his studies, he focused on several aspects of chromatin research like chromatin remodelling assays, DNA repair and proteomics analysis of chromatin complexes.
    • Ludwig Maximilians University Munich (LMU), Germany PostDoc
      Biography
        Since 2013, Dr. Andreas Schmidt is employed as PostDoc for development of protocols for proteomics analysis and proteomics data analysis in the group of Prof. A. Imhof at the Department of Molecular Biology of the Medical Faculty of the Ludwig-Maximilians University of Munich. The focus of his work is development of data independent acquisition methods for quantitation of proteins and protein modifications as well as improving the data analysis of data-independent mass spectrometry data. For his PhD studies and first PostDoc, he joined the mass spectrometry unit of K. Mechtler and Prof. G. Ammerer at the Institute for Molecular Pathology, studying phosphorylation of arginine as stress-induced protein tag in bacteria. In collaboration with the group of T. Clausen, they described the protein arginine kinase and improved enrichment and identification of peptides with this modification. He received a diploma degree in chemistry from the University of Leipzig, where he performed my thesis in the junior research group of A. Sinz on structural investigation of the Calmodulin/Adenylyl cyclase VIII complex using chemical crosslinking and mass spectrometry.

      Abstract
      DATE: November 23rd, 2016
      TIME: 8:00AM PT, 11:00AM ET


      The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. The nucleosomes together with the non-histone proteins define a stable chromatin structure. Despite its stability, this structure is disassembled and reassembled during DNA replication, repair, recombination or transcription. During all those processes, defined chromatin regions become accessible to be bound by the required factors, resulting in extensive nucleosome turnover at given genomic loci. The dual nature of chromatin requires a continuous interplay between stable and dynamic structures, which has to be coordinated at the molecular level to maintain the epigenetic information stored in the chromatin structure.
      Despite the biological relevance of these processes, little is known about the order of chromatin assembly steps, the molecular mechanisms that coordinate the required cellular machinery in time and the quality control of this assembly. 
      To address these questions, our lab uses an in-vitro system that resembles the formation of chromatin on double-stranded DNA. This in-vitro system not only enables us to dissect critical steps of assembly but also to verify predictions we make based on proteomics analysis of captured nascent chromatin (NCC) in living cells.
      In order to study the dynamics of chromatin-bound proteins, we applied label free quantitative SWATH-MS (sequential window acquisition of all theoretical fragment-ion spectra) at different assembly times, allowing us to describe distinct aspects of chromatin assembly such as the appearance and disappearance of histone modifications, the levels of histone chaperones, the activity of histone writers/erasers or the concentration of distinct DNA-binding factors. Based on these results, we have accomplished a classification of chromatin factors into functional groups depending on their binding kinetics.

      Learning Objective 1: Develop an experimental outline to study chromatin assembly in vitro and in vivo with quantitative proteomics

      Learning Objective 2: Establish a SWATH proteomics pipeline combined with statistical data analysis to determine protein binding kinetics

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