Genoproteomics: An Integrated Approach to Analyze the Protein Fraction of Mouse Milk
1,2Bellier S., 1Boumahrou N., 1,3Andrei S., 1Miranda G., 1Beauvallet C., 1Bevilacqua C., 1Zalachas E., 4Henry C., 2Panthier J.J. and 1Martin P. 1 Génomique & Physiologie de la Lactation, INRA Jouy en Josas, France 2 Génétique moléculaire et cellulaire, UMR 955 INRA-ENVA, Maisons-Alfort, France 3 Universitatea de Stiinte Agricole si Medicina Veterinara Cluj-Napoca,Romania 4 Biochimie Bactérienne, PAPSS, INRA Jouy en Josas, France
Mouse milk proteins have been the subject of many studies so far.
Surprisingly, to our knowledge, none of these studies provides a clear
enough and complete description of the main protein fraction of mouse
milk. To overcome such an unexpected deficiency we have undertaken to
develop a reliable technique with a high resolving power to separate,
identify and characterize the major mouse milk proteins. This approach
was achieved through the study of three mouse strains (C57BL/6, PWK and
SEG) representative of mouse species (Mus m. domesticus, Mus m.
musculus and Mus spretus, respectively) used in cross-experiments for
genetic mapping purpose.
C57BL/6 skimmed milk was first fractionated by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) and the protein content of each of the nine major peaks obtained was further analyzed by SDS-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE). Protein bands extracted from the gel were digested by trypsin and the peptides were analyzed on a Matrix–Assisted Laser Desorption/Ionization-Time of Flight (MALDI-Tof) mass spectrometer. Proteins were subsequently identified by Peptide Mass Fingerprinting (PMF), matching their peptide masses (tryptic digests) to the theoretical mass list generated from databases. This approach has allowed the identification of eight major mouse milk proteins: kbge and as1 caseins, lactoferrin (Lf), serum albumin (SA) and the whey acidic protein (WAP). In the three mouse strains, SDS-PAGE revealed the occurrence of a minor as1-casein isoform, characterized after cDNA cloning and sequencing as a splicing variant.
Then, RP-HPLC profiles, mono and two-dimensional electrophoresis patterns of C57BL/6 mouse milk proteins were compared with those obtained starting from milk of PWK and SEG strains. This comparative study revealed that only five of the eight major proteins (Lf, SA and k-, g-, e-caseins) had the same retention time (RP-HPLC) and electrophoresis mobility between strains. The other three proteins (b-, as1-caseins and WAP) exhibited different behaviours depending on the mouse species. Mutations responsible for these differences have been identified after cDNA cloning and sequencing. These results were confirmed at the protein level using high accuracy mass measurements on an orbitrap mass spectrometer (LTQ-orbitrap).