A Method for the Quantitative Analysis of Protein-Protein Interactions In Vivo

by Nils Arne Rall
Doctoral thesis
Date of Examination:2016-03-22
Date of issue:2016-04-13
Advisor:Prof. Dr. Heinz Neumann
Referee:Prof. Dr. Heinz Neumann
Referee:Prof. Dr. Henning Urlaub
Persistent Address: http://dx.doi.org/10.53846/goediss-5609

 

 

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Abstract

English

The function and activity of proteins is often modulated by other proteins they interact with. To understand cellular behaviour at the system level, a complete description of interactions among different proteins and the dynamic nature of these interactions is required.  Identification and quantification of proteins based on measured peptide intensities represents the highest standard for an unbiased and definite determination of protein interaction partners.  Inspired by the display of effectiveness of pBPA in vivo crosslinking in elucidating the mechanistic details of chromatin condensation in S. cerevisiae (Wilkins et al., 2014), the research objective of this PhD thesis was the development of a method for the quantitative identification of histone interaction partners for further decoding of chromatin structure dynamics. First, the effectiveness of pBPA in vivo crosslinking for capturing histone-protein interactions in living yeast cells was successfully confirmed. Secondly, an immunoprecipitation protocol for pulldown of HA-tagged histone pBPA mutants and their UV-induced crosslink products was established and adapted to SILAC conditions. Thirdly, initial SILAC analyses were performed and optimized until reproducible results of increased quality were obtained. Optimization led to the identification of numerous chromatin-related proteins, including a confirmed interaction partner. 
Keywords: Genetic code expansion; SILAC; Mass spectrometry; Chromatin; Histones; Crosslinking; In vivo
 
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