Christine Gauss

Analysis of the mouse proteome. (I) Brain proteins: Separation by two‐dimensional electrophoresis and identification by mass spectrometry and genetic variation

The total protein of the mouse brain was fractionated into three fractions, supernatant, pellet extract and rest pellet suspension, by a procedure that avoids any loss of groups or classes of proteins. The supernatant proteins were resolved to a maximum by large‐gel two‐dimensional electrophoresis. Two‐dimensional patterns from ten individual mice of the commonly used inbred strain C57BL/6 (species: Mus musculus) were prepared. The master pattern was subjected to densitometry, computer‐assisted image analysis and treatment with our spot detection program. The resulting two‐dimensional pattern, a standard pattern for mouse brain supernatant proteins, was divided into 40 squares, calibrated, and specified by providing each spot with a number. The complete pattern and each of the 40 squares are shown in our homepage (http://www.charite.de/humangenetik). The standard pattern comprises 8767 protein spots. To identify the proteins known so far in the brain fraction investigated, a first set of 200 spots was analyzed by matrix‐assisted laser desorption/ionization ‐ mass spectrometry (MALDI‐MS) after in‐gel digestion. By screening protein databases 115 spots were identified; by extending the analysis to selected, genetically variant protein spots, 166 spots (including some spot series) were identified in total. This number was increased to 331 by adding protein spots identified indirectly by a genetic approach. By comparing the two‐dimensional patterns from C57BL/6 mice with those of another mouse species (Mus spretus), more than 1000 genetically variant spots were detected. The genetic analysis allowed us to recognize spot families, i.e., protein spots that represent the same protein but that are post‐translationally modified. If some members of the family were identified, the whole family was considered as being identified. Spot families were investigated in more detail, and interpreted as the result of protein modification or degradation. Genetic analysis led to the interesting finding that the size of spot families, i.e., the extent of modification or degradation of a protein, can be genetically determined. The investigation presented is a first step towards a systematic analysis of the proteome of the mouse. Proteome analysis was shown to become more efficient, and, at the same time, linked to the genome, by combining protein analytical and genetic methods.

Technology development at the interface of proteome research and genomics: Mapping nonpolymorphic proteins on the physical map of mouse chromosomes

Data obtained from protein spots by peptide mass fingerprinting are used to identify the corresponding genes in sequence databases. The relevant cDNAs are obtained as clones from the Integrated Molecular Analysis of Genome Expression (I.M.A.G.E.) consortium. Mapping of I.M.A.G.E. clones is performed in two steps: first, cDNA clones are hybridized against a 10‐hit genomic mouse bacterial artificial chromosome (BAC) library. Second, interspersed repetitive sequence polymerase chain reaction (IRS‐PCR) using a single primer directed against the mouse B1 repeat element is performed on BACs. As each cDNA detects several BACs, and each individual BAC has a 50% chance to recover an IRS‐PCR fragment, the majority of cDNAs produce at least a single IRS‐PCR fragment. Individual IRS fragments are hybridized against high‐density spotted filter grids containing the three‐dimensional permutated pools of yeast artificial chromosome (YAC) library resources that are currently being used to construct a physical map of the mouse genome. IRS fragments that hybridize to YAC clones already placed into contigs immediately provide highly precise map positions. This technology therefore is able to draw links between proteins detected by 2‐D gel electrophoresis and the corresponding gene loci in the mouse genome.