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Proteomic tools to characterize the protein fraction of Equidae milk.
Abstract: 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. kappa-, alpha(s1)-, alpha(s2)-, and beta-casein, lysozyme C, alpha-lactalbumin and beta-lactoglobulin I and II were thus identified. alpha(s1) and beta-caseins displayed polymorphic patterns, probably due to alternative splicing processes leading to casual exon skipping events involving exons 7 and 14 in alpha(s1)-casein and exon 5 in beta-casein. Edman N-terminal microsequencing over 35 amino acid residues, for pony alpha(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.
Publication Date: 2004-07-27 PubMed ID: 15274143DOI: 10.1002/pmic.200300765Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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This research successfully identified and partially characterized the main protein components in pony mare’s milk using proteomic tools, highlighting the similarities between Equidae and human milk in protein content.
Research Methodology
- The study used skimmed pony mare’s milk, where the protein content was obtained through reversed phase-high-performance liquid chromatography (RP-HPLC) or a bi-dimensional separation technique combining RP-HPLC and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE).
- These obtained protein fractions were further analyzed with Edman N-terminal microsequencing and mass determination, both with and without tryptic digestion, with a matrix-assisted laser desorption/ionization-time of flight spectrometer.
- Upon acquiring information regarding the sequence and molecular mass, identifications of proteins were made by searching protein databases using homology or pattern research algorithms.
Research Findings
- The proteomic tools used in the research proved efficient and rapid in identifying the principal proteins in pony mare’s milk. These included kappa-, alpha(s1)-, alpha(s2)-, and beta-casein, lysozyme C, alpha-lactalbumin, and beta-lactoglobulin I and II.
- Polymporphism patterns were observed in alpha(s1) and beta-caseins, likely caused by alternative splicing processes that result in casual exon skipping events with exons 7 and 14 in alpha(s1)-casein and exon 5 in beta-casein.
- Edman N-terminal microsequencing of over 35 amino acids residues for alpha(s1)-casein indicated a splicing pattern in Equidae similar to that noted in rodents, specifically the regular outsplicing of exon 5.
Comparative Study and Conclusion
- The research also compared the SDS-PAGE and RP-HPLC patterns of mare’s milk with those obtained from cow, goat, and human milks, including the relative levels of caseins and major whey proteins.
- Results emphasized that in terms of casein and lysozyme C contents, as well as the casein-to-whey protein ratio, Equidae milk closely resembles human breast milk than milk from bovines and caprines.
Cite This Article
APA
Miranda G, Mahé MF, Leroux C, Martin P.
(2004).
Proteomic tools to characterize the protein fraction of Equidae milk.
Proteomics, 4(8), 2496-2509.
https://doi.org/10.1002/pmic.200300765 Publication
Researcher Affiliations
- Laboratoire de Biochimie et Structure des Protéines, Institut National de la Recherche Agronomique, Jouy-en-Josas, France. guy.miranda@jouy.inra.fr
MeSH Terms
- Amino Acid Sequence
- Animals
- Cattle
- Chromatography, High Pressure Liquid / methods
- Electrophoresis, Gel, Two-Dimensional
- Electrophoresis, Polyacrylamide Gel
- Female
- Goats
- Horses
- Humans
- Milk / chemistry
- Milk Proteins / analysis
- Milk Proteins / genetics
- Molecular Sequence Data
- Molecular Weight
- Peptides / analysis
- Proteomics / methods
- Sequence Alignment
Citations
This article has been cited 14 times.- Reichler SJ, Orta-Ramirez A, Martin NH, Wiedmann M. Culture-independent bacterial cell extraction from fluid milk and oat-based beverage for basic qualitative microscopy. JDS Commun 2023 Mar;4(2):65-69.
- Zhao S, Pan F, Cai S, Yi J, Zhou L, Liu Z. Secrets behind Protein Sequences: Unveiling the Potential Reasons for Varying Allergenicity Caused by Caseins from Cows, Goats, Camels, and Mares Based on Bioinformatics Analyses. Int J Mol Sci 2023 Jan 27;24(3).
- Zhang X, Jiang G, Ji C, Fan Z, Ge S, Li H, Wang Y, Lv X, Zhao F. Comparative Whey Proteome Profiling of Donkey Milk With Human and Cow Milk. Front Nutr 2022;9:911454.
- Raux A, Bichon E, Benedetto A, Pezzolato M, Bozzetta E, Le Bizec B, Dervilly G. The Promise and Challenges of Determining Recombinant Bovine Growth Hormone in Milk. Foods 2022 Jan 20;11(3).
- Wang Y, Miao X, Zhao Z, Wang Y, Li S, Wang C. Transcriptome Atlas of 16 Donkey Tissues. Front Genet 2021;12:682734.
- He M, Nie X, Wang H, Yan S, Zhang Y. Effects of a High-Grain Diet With a Buffering Agent on Milk Protein Synthesis in Lactating Goats. Front Vet Sci 2021;8:696703.
- Miranda G, Bianchi L, Krupova Z, Trossat P, Martin P. An improved LC-MS method to profile molecular diversity and quantify the six main bovine milk proteins, including genetic and splicing variants as well as post-translationally modified isoforms. Food Chem X 2020 Mar 30;5:100080.
- Ryskaliyeva A, Henry C, Miranda G, Faye B, Konuspayeva G, Martin P. Alternative splicing events expand molecular diversity of camel CSN1S2 increasing its ability to generate potentially bioactive peptides. Sci Rep 2019 Mar 27;9(1):5243.
- Ryskaliyeva A, Henry C, Miranda G, Faye B, Konuspayeva G, Martin P. Combining different proteomic approaches to resolve complexity of the milk protein fraction of dromedary, Bactrian camels and hybrids, from different regions of Kazakhstan. PLoS One 2018;13(5):e0197026.
- Polidori P, Vincenzetti S. Use of Donkey Milk in Children with Cow's Milk Protein Allergy. Foods 2013 May 6;2(2):151-159.
- Islam MA, Ekeberg D, Rukke EO, Vegarud GE. Ex vivo Digestion of Milk from Red Chittagong Cattle Focusing Proteolysis and Lipolysis. Asian-Australas J Anim Sci 2015 Apr;28(4):559-67.
- Shokrollahi B, Choi JY, Won M, Kim ET, Lee SE, Ham JS. Koumiss (Fermented Mare's Milk) as a Functional Food: Bioactive Proteins, Peptides, and Future Perspectives. Foods 2025 Nov 18;14(22).
- Blanco-Doval A, Barron LJR, Aldai N. Nutritional Quality and Socio-Ecological Benefits of Mare Milk Produced under Grazing Management. Foods 2024 May 4;13(9).
- Mayerl CJ, German RZ. Evolution, diversification and function of the maternal-infant dyad in mammalian feeding. Philos Trans R Soc Lond B Biol Sci 2023 Dec 4;378(1891):20220554.
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