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Proteomic tools to characterize the protein fraction of Equidae milk
Author(s) -
Miranda Guy,
Mahé MarieFrançoise,
Leroux Christine,
Martin Patrice
Publication year - 2004
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200300765
Subject(s) - edman degradation , chromatography , chemistry , casein , biology , peptide sequence , biochemistry , microbiology and biotechnology , gene
The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase‐high‐performance liquid chromatography (RP‐HPLC) on a C4 column or a bi‐dimensional separation technique coupling RP‐HPLC in the first dimension and sodium dodecyl sulfate‐polyacrylamide electrophoresis (SDS‐PAGE) in the second dimension (two‐dimensional RP‐HPLC/SDS‐PAGE). The fractions thus obtained were analyzed by Edman N ‐terminal microsequencing and mass determination, with or without tryptic digestion, on a matrix‐assisted laser desorption/ionization‐time of flight spectrometer. Based on the sequence and molecular mass information obtained, identifications were achieved through a protein database search using homology or pattern research algorithms. This methodological approach was shown to be rapid, efficient and reliable in identifying the principal proteins in pony mare's milk. κ‐, α s1 ‐, α s2 ‐, and β‐casein, lysozyme C, α‐lactalbumin and β‐lactoglobulin I and II were thus identified. α s1 and β‐caseins displayed polymorphic patterns, probably due to alternative splicing processes leading to casual exon skipping events involving exons 7 and 14 in α s1 ‐casein and exon 5 in β‐casein. Edman N ‐terminal microsequencing over 35 amino acid residues, for pony α s1 ‐casein, clearly demonstrated the occurrence, in Equidae , of a splicing pattern similar to that reported in rodents, characterized by the constitutive outsplicing of exon 5. Pony mare's milk SDS‐PAGE and RP‐HPLC patterns were compared with those obtained for other milks (cow, goat and human), as were the relative levels of caseins and major whey proteins in these milks. Our results provide further evidence to support the notion that Equidae milk is closer to human breast milk than milk from bovine and caprine with respect to the casein and lysozyme C contents and casein/whey proteins ratio.