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MPI für Biochemie  

Membrane Biochemistry
Dieter Oesterhelt

Research Projects - Frank Siedler

 

Our Mission

Within the department of "Membrane Biochemistry", it is our responsibility to deliver qualitative and quantitative knowledge about proteins, lipids, and small metabolites in halophilic archaea. By using various mass spectrometry approaches, hard experimental data are produced for the use in a systems biology context.

Proteomics

2D gel
Focus: The cytosolic protein inventory of Halobacterium salinarum.
By using an optimized 2D electrophoresis approach combined with semi-automatic trypsin digestion, robotic sample handling, and MALDI-PMF we were able to identify about half of the theoretical cytosolic proteome.
A reference map for further studies was established.

 

Small Proteins
Focus: The extention of proteomics technology to cover problematic proteins (e.g. small proteins).
By using size exclusion filtering, a 6-fold enhancement of the identification rate for proteins of mass below 20kDa could be reached.

Workflow Napha
Focus: The cytosolic protein inventory of Natronomonas pharaonis.
Different sample preparations in combination with Nano-LC-MS/MS technology allowed for the identification of about 60% of the organisms theoretical cytosolic proteome. In comparison to Halobacterium salinarum, important conclusions about the genetic variability in halophilic archaea were possible.

Permeases
Focus: The membrane proteome of Halobacterium salinarum.
By in depth optimization of all parameters it could be shown that 2D electrophoresis is in general not suitable for the analysis of complete membrane proteomes. The solution is a geLC-MS/MS approach, which allowed for the identification of 20% of the predicted TM proteins.

16BAC/SDS - DIGE
Focus: Quantitative membrane proteomics of Halobacterium salinarum.
To compare two growth states that strongly affect the energy supply of the cells (aerobic versus anaerobic/phototrophic conditions) a gel based quantitation approach (DIGE) and a MS based approach (ICPL) were used in combination. A total of 155 membrane proteins could be quantified and allowed for important conclusions about the organisms metabolism.

N-terms methods
Focus: Identification of N-terminal peptides in Halobacterium salinarum and Natronomonas pharaonis.
Specific (COFRADIC, SCX) and non-specific proteomic approaches were used to gain information about protein N-termini. Besides the direct support for our partner group "Bioinformatics" (start codon assignment), this dataset represents a valuable ressource for further studies (see "N-terminal protein maturation in archaea").

N-terms results
Focus: N-terminal protein maturation in archaea.
Based on the proteomical identification of 837 protein N-termini in Halobacterium salinarum and Natronomonas pharaonis, pattern of N-terminal methionine cleavage and/or acetylation were analyzed. Results allow for conclusions in relation to bacteria and eukarya.

Htr14 methylation
Focus: Transducer deamidation and methylation in Halobacterium salinarum.
Based on a geLC-MS/MS approach, 19 methylation sites in 10 of the 12 theoretically predicted membrane-spanning sensory transducers were identified. Important conclusions can be drawn about signal adaptation in archaea.

Lipid Analysis

lipids
The mechanisms of lipid ana- and catabolic processes are investigated. Lipid specific protocols have been established for chromatography (HPLC), cleavage and mass spectrometric identification and quantitation via GC/MS.

Metabolites

metabolites
To support metabolite related systems biology, the quantitation of small molecules via GC/MS was optimized. The establishment of complementary LC/MS-based methods has been started.