Genetic analysis of the mouse brain proteome

Abstract

Proteome analysis is a fundamental step in systematic functional genomics. Here we have resolved 8,767 proteins from the mouse brain proteome by large-gel two-dimensional electrophoresis. We detected 1,324 polymorphic proteins from the European collaborative interspecific backcross. Of these, we mapped 665 proteins genetically and identified 466 proteins by mass spectrometry. Qualitatively polymorphic proteins, to 96%, reflect changes in conformation and/or mass. Quantitatively polymorphic proteins show a high frequency (73%) of allele-specific transmission in codominant heterozygotes. Variations in protein isoforms and protein quantity often mapped to chromosomal positions different from that of the structural gene, indicating that single proteins may act as polygenic traits. Genetic analysis of proteomes may detect the types of polymorphism that are most relevant in disease-association studies.

Acute and long-term proteome changes induced by oxidative stress in the developing brain

“…40,41 In short, frozen brain tissue, 1.6 parts v/w of buffer 1 (0.11 M CHAPS, 50 mM TRIZMA s Base (Sigma-Aldrich, Steinheim, Germany), 50 mM KCl and 20% w/v glycerol at pH 7.5), 0.08 parts of protease inhibitor solution I (1 Completet tablet (Roche Applied Science, Mannheim, Germany) dissolved in 2 ml of buffer 1) and 0.02 parts of protease inhibitor solution II (1.4 mM pepstatin A and 1 mM phenylmethylsulfonyl fluoride in ethanol) were ground to fine powder in a mortar precooled in liquid nitrogen. The tissue powder was transferred into a 2 ml tube, quickly thawed and supplied with an average number of 11 glass beads (0.034 units of glass beads per combined weight of tissue, buffers and inhibitors in mg).…”

“…40,41 The gel format was 40 cm (isoelectric focussing) Â 30 cm (SDS-PAGE) Â 0.75 mm. For isoelectric focusing (IEF) using the mobile ampholyte technique, we applied 6 ml (B20 mg/ ml) protein extract of each sample to the anodic end of an IEF-gel and used a carrier ampholyte mixture to establish a pH gradient in a range from 3 to 10.…”

“…Proteins were visualized in SDS-PAGE polyacrylamide gels by high sensitivity silver staining. 41 2-DE gels were evaluated visually by a trained observer on a light box (Biotec-Fischer, Reiskirchen, Germany). Spot changes were considered with respect to presence/absence, quantitative variation and altered mobility.…”

“…SDS-PAGE gels were stained with Coomassie blue staining as previously described. 40,41 Protein carbonyls were also identified following 2-DE through a modified protocol. Prior to derivatization, brain proteins were separated in 15 cm IEF gels according to their isoelectric point using the mobile ampholyte technique described above.…”

PROTEOMER: A workflow‐optimized laboratory information management system for 2‐D electrophoresis‐centered proteomics

“…For this purpose, we semi-automatically compared the protein expression patterns of two mouse strains (Mus musculus and Mus spretus) and their F1-generation in three different tissues: brain [39], liver, and heart. The 2-D gels were originally generated in the context of different projects to answer distinct biological questions.…”