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Biochimie open2017; 4; 61-72; doi: 10.1016/j.biopen.2017.02.004

Purified horse milk exosomes contain an unpredictable small number of major proteins.

Abstract: Exosomes are 40-100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel filtration. Individual preparations demonstrated different profiles of gel filtration showing well or bad separation of exosome peaks and one or two peaks of co-isolating proteins and their complexes. According to the electron microscopy, well purified exosomes displayed a typical exosome-like size (30-100 nm) and morphology. It was shown that exosomes may have several different biological functions, but detection of their biological functions may vary significantly depending on the presence of exosome contaminating proteins and proteins directly into exosomes. Exosome proteins were identified before and after gel filtration by MALDI MS and MS/MS spectrometry of protein tryptic hydrolyzates derived by SDS PAGE and 2D electrophoresis. The results of protein identification were unexpected: one or two peaks co-isolating proteins after gel-filtration mainly contained kappa-, beta-, alpha-S1-caseins and its precursors, but these proteins were not found in well-purified exosomes. Well-purified exosomes contained from five to eight different major proteins: CD81, CD63 receptors, beta-lactoglobulin and lactadherin were common to all preparations, while actin, butyrophilin, lactoferrin, and xanthine dehydrogenase were found only in some of them. The article describes the morphology and the protein content of major horse milk exosomes for the first time. Our results on the decrease of major protein number identified in exosomal preparations after gel filtration may be important to the studies of biological functions of pure exosomes.
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Publication Date: 2017-03-01 PubMed ID: 29450143PubMed Central: PMC5801828DOI: 10.1016/j.biopen.2017.02.004Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article discusses the extraction, purification, and protein content analysis of exosomes found in horse milk, providing an overview of the proteins these nanoparticles contain and highlighting the potential importance of these findings in understanding the biological functions of exosomes.

Research Methodology

  • The study begins by explaining that exosomes are nanovesicles ranging around 40-100 nm in size that carry RNA and various proteins within them.
  • They are critical for intracellular communication and immune function due to the proteins, lipids, mRNAs, and microRNAs they contain.
  • Exosomes are generally acquired through a combination of centrifugation and ultracentrifugation techniques, and are known to contain anywhere from a few dozen to thousands of different proteins, according to previously published data.
  • In this study, initial exosome preparations were obtained from the milk of eighteen horses.

Purification and Analysis

  • The exosome preparations were further purified using FPLC gel filtration, a method used to separate and purify proteins according to their size.
  • Different filtration profiles were displayed by individual preparations, with some showing clear separation of exosome peaks and others not.
  • The study examined the samples using electron microscopy and found that well-purified exosomes exhibited the typical size and morphology expected of exosomes.
  • The study confirmed that exosomes can perform various biological functions, however, the detection of these functions can significantly vary based on the presence of exosome contaminating proteins and proteins directly into exosomes.

Protein Identification

  • The next phase of the research involved identifying the proteins present in the exosomes. This was done both before and after gel filtration using MALDI mass spectrometry and tandem mass spectrometry of protein tryptic hydrolyzates derived by SDS PAGE and 2D electrophoresis.
  • The results were unexpected, with one or two protein peaks isolating after gel-filtration mainly containing kappa-, beta-, alpha-S1-caseins and its precursors, however, these proteins were not discovered in well-purified exosomes.
  • Instead, five to eight different major proteins were found in the well-purified samples.

Conclusion

  • This is the first report detailing the morphology and protein content of major horse milk exosomes. The researchers pointed out that their results can play a significant role in future studies of the biological functions of pure exosomes, particularly due to the decrease of major protein number identified in exosomal preparations after gel filtration.

Cite This Article

APA
Sedykh SE, Purvinish LV, Monogarov AS, Burkova EE, Grigor'eva AE, Bulgakov DV, Dmitrenok PS, Vlassov VV, Ryabchikova EI, Nevinsky GA. (2017). Purified horse milk exosomes contain an unpredictable small number of major proteins. Biochim Open, 4, 61-72. https://doi.org/10.1016/j.biopen.2017.02.004

Publication

Biochimie open
ISSN: 2214-0085
NlmUniqueID: 101706117
Country: Netherlands
Language: English
Volume: 4
Pages: 61-72

Researcher Affiliations

Sedykh, Sergey E
  • SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., Novosibirsk 630090, Russia.
  • Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russia.
Purvinish, Lada V
  • SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., Novosibirsk 630090, Russia.
  • Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russia.
Monogarov, Artem S
  • SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., Novosibirsk 630090, Russia.
Burkova, Evgeniya E
  • SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., Novosibirsk 630090, Russia.
  • Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russia.
Grigor'eva, Alina E
  • SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., Novosibirsk 630090, Russia.
Bulgakov, Dmitrii V
  • Institute of Biology and Soil, Far East Division, Russian Academy of Sciences, Vladivostok 690022, Russia.
Dmitrenok, Pavel S
  • Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences, Vladivostok 690022, Russia.
Vlassov, Valentin V
  • SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., Novosibirsk 630090, Russia.
  • Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russia.
Ryabchikova, Elena I
  • SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., Novosibirsk 630090, Russia.
  • Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russia.
Nevinsky, Georgy A
  • SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., Novosibirsk 630090, Russia.
  • Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russia.

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Citations

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