Project description:Major Urinary Proteins (MUPs) from different inbred strains of mouse have been analysed by high-resolution ion-exchange chromatography and mass spectrometry. MUPs from six strains were resolved chromatographically into four major protein peaks which characterized two distinct phenotypes, typified by the profiles obtained from the Balb/c and C57BL/6 inbred strains. A combination of ion-exchange chromatography and electrospray ionization mass spectrometry analysis of the MUPs from each strain identified five proteins, only one of which was common to both strains. The charge and mass data, together with N-terminal sequence analyses, were correlated with the masses of the proteins inferred from published cDNA sequences. Several members of the family of MUP sequences differ in only four positions, and in some circumstances the substitutions elicit a minimal change in protein mass (Lys/Gln; Lys/Glu). Peptide mapping with endopeptidase Lys-C, followed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry permitted identification of new MUPs that were correlated with partial cDNA sequence data. In the two strains there are at least 13 different MUPs, either observed or predicted, indicating the heterogeneity of expression of this group of proteins.
Project description:BACKGROUND: Due to their high level of genotypic and phenotypic variability, Mus spretus strains were introduced in laboratories to investigate the genetic determinism of complex phenotypes including quantitative trait loci. Mus spretus diverged from Mus musculus around 2.5 million years ago and exhibits on average a single nucleotide polymorphism (SNP) in every 100 base pairs when compared with any of the classical laboratory strains. A genoproteomic approach was used to assess polymorphism of the major milk proteins between SEG/Pas and C57BL/6J, two inbred strains of mice representative of Mus spretus and Mus musculus species, respectively. RESULTS: The milk protein concentration was dramatically reduced in the SEG/Pas strain by comparison with the C57BL/6J strain (34 ± 9 g/L vs. 125 ± 12 g/L, respectively). Nine major proteins were identified in both milks using RP-HPLC, bi-dimensional electrophoresis and MALDI-Tof mass spectrometry. Two caseins (? and ?s1) and the whey acidic protein (WAP), showed distinct chromatographic and electrophoresis behaviours. These differences were partly explained by the occurrence of amino acid substitutions and splicing variants revealed by cDNA sequencing. A total of 34 SNPs were identified in the coding and 3'untranslated regions of the SEG/Pas Csn1s1 (11), Csn2 (7) and Wap (8) genes. In addition, a 3 nucleotide deletion leading to the loss of a serine residue at position 93 was found in the SEG/Pas Wap gene. CONCLUSION: SNP frequencies found in three milk protein-encoding genes between Mus spretus and Mus musculus is twice the values previously reported at the whole genome level. However, the protein structure and post-translational modifications seem not to be affected by SNPs characterized in our study. Splicing mechanisms (cryptic splice site usage, exon skipping, error-prone junction sequence), already identified in casein genes from other species, likely explain the existence of multiple ?s1-casein isoforms both in SEG/Pas and C57BL/6J strains. Finally, we propose a possible mechanism by which the hallmark tandem duplication of a 18-nt exon (14 copies) may have occurred in the mouse genome.