The cytosolic proteome of Halobacterium salinarum
The membrane proteome of Halobacterium salinarum
Cytosolic proteome publication:
A. Tebbe, Ch. Klein , B. Bisle, F. Siedler, B. Scheffer, C. Garcia-Rizo, J. Wolfertz, V. Hickmann, F. Pfeiffer, D. Oesterhelt
Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation.
Proteomics 5, 168-179 (2005
Membrane proteome publication:
Ch. Klein , C. Garcia-Rizo, B. Bisle, B. Scheffer, H. Zischka, F. Pfeiffer, F. Siedler, D. Oesterhelt
The membrane proteome of Halobacterium salinarum.
Proteomics 5, 180-197 (2005)
We attempt to identify the whole protein inventory of Halobacterium salinarum. Proteins are separated by 2D gel electrophoresis resulting in a set of more than 1,000 distinct protein spots (Fig.1).
Protein spots are picked out of the gel and digested into peptide fragments using trypsin, a digestive enzyme from the pancreas. The resulting set of fragments can be used as "fingerprint" to identify the protein. The fragment masses are determined using a mass spectrometer. The experimentally determined masses are then compared to the theoretically computed masses using the MASCOT program. The protein has been identified when a sufficient number of matches is found. To avoid errors, we consider a protein identified only when it comes up with a very high score.
So far we have been able to identify more than 900 proteins out of 2784 open reading frames (ORFs) believed to code for real proteins. The database of all proteins from Halobacterium is derived from the [»] complete genome sequence which has been determined in our department.
But instead of just providing proteomics with the necessary tools, both approaches, genomics and proteomics, suddenly find themselves in a classical yin-yang situation: proteomics needs genome-derived information in form of the database. Genomics experimentally creates the raw DNA sequence. All the interpretion of the sequence depends on computer programs and bioinformatics. Such programs, like gene finders (also called ORF predictors) may create incorrect results. This is especially evident in the genome of H. salinarum where by far too many ORFs exist. Questions like "Which of the ORFs code for real proteins?" and "Where does the protein actually start?" can typically be answered by proteomics. These experimental results are then used to update the genome annotation and thus to create a more correct protein sequence database.
Moreover, informations about the existance of proteins known to be of high importance for certain metabolic pathways are valuable. Once the protein inventory is complete, the dynamic proteomics work will get more into focus. Questions like "How does the organism react on the protein level on changes in the environment like salt concentration or light conditions?" will be of future interest.