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 tha t avoids any loss of groups or classes of proteins. The supernatant protein s were resolved to a maximum by large-gel two-dimensional electrophoresis. Two-dimensional patterns from ten individual mice of the commonly used inbr ed strain C57BL/6 (species: Mus musculus) were prepared. The master pattern was subjected to densitometry, computer-assisted image analysis and treatm ent 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 lase r desorption/ionization - mass spectrometry (MALDI-MS) after in-gel digesti on. By screening protein databases 115 spots were identified; by extending the analysis to selected, genetically variant protein spots, 166 spots (inc luding some spot series) were identified in total. This number was increase d to 331 by adding protein spots identified indirectly by a genetic approac h. By comparing the two-dimensional patterns from C57BL/6 mice with those o f another mouse species (Mus spretus), more than 1000 genetically variant s pots were detected. The genetic analysis allowed us to recognize spot famil ies, i.e., protein spots that represent the same protein but that are post- translationally modified. If some members of the family were identified, th e whole family was considered as being identified. Spot families were inves tigated in more detail, and interpreted as the result of protein modificati on 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. Prot eome analysis was shown to become more efficient, and, at the same time, li nked to the genome, by combining protein analytical and genetic methods.