TY  - GEN
Y1  - 2012/11/08/
KW  - Proteomics
Mass spectrometry
Secretome analysis
Protein synthesis
Macrophages
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/13972/
AV  - public
ID  - heidok13972
N2  - Transcription and translation are precisely-regulated processes that enable a cell to react to environmental stimuli by the production of appropriate sets of proteins. In order to analyze gene expression regulation, two novel methods have been developed that facilitate affinity purification approaches in combination with quantitative mass spectrometry.
First, the concepts underlying transcriptional control are investigated by identifying proteins binding to cis-regulatory modules (CRMs) in a sequence-specific manner. The method facilitated the determination of proteins specifically binding to DNA sequences with a length of 500 base pairs. Quantitative comparison of the binding pattern of three CRMs active in Drosophila melanogaster muscle development revealed 72 candidates potentially regulating the activity of these CRMs among thousands of proteins that interact non-specifically. In vivo validation of biological activity of several candidates is in progress and will probably reveal new circuits of Drosophila melanogaster muscle development.
Second, a novel approach to specifically enrich and quantify newly synthesized proteins by combining click-chemistry and pulsed SILAC labeling was developed. This method was introduced as a useful tool to study protein synthesis and the sensitive detection of rapid response to cellular stimulation.
In addition, the method was adapted to the selective and precise quantification of secreted proteins. This important subset of mammalian proteins is currently understudied because of technical limitations in the detection of low-abundant proteins against a background of serum. In-depth and differential secretome analysis of various cell lines and primary cells revealed, e.g., profound effects of serum starvation on secretome composition. Moreover, a unique application studying the kinetics of protein secretion was introduced.
The approach will have broad implications in studying the responsiveness of cells grown under optimal conditions.
Finally, in combination with RNA and protein abundance measurements, the developed approaches were used to investigate regulatory mechanism establishing response programs in lipopolysaccharides stimulated mouse macrophages with temporal resolution. These data for the first time provide a comprehensive view on the kinetics of macrophage activation. Transcriptional, translational and localization regulation was distinguished and starting points for further investigation of these mechanisms were proposed.

TI  - Quantitative proteomics of transcriptional and translational regulation
A1  - Eichelbaum, Katrin
ER  -