TY - JOUR

T1 - Analysis of Proteins and Peptides of Highly Purified CD9+ and CD63+ Horse Milk Exosomes Isolated by Affinity Chromatography

AU - Sedykh, Sergey E

AU - Purvinsh, Lada V

AU - Burkova, Evgeniya E

AU - Dmitrenok, Pavel S

AU - Ryabchikova, Elena I

AU - Nevinsky, Georgy A

N1 - This research was funded by the RSF, grant number 18-74-10055 to Sergey Sedykh and 0245-2021-0009 (121031300041-4) to Georgy Nevinsky (MALDI analysis).

PY - 2022/12/17

Y1 - 2022/12/17

N2 - Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we isolated highly purified vesicle preparations from 18 horse milk samples. Gel filtration of the initial preparations allowed us to remove co-isolating proteins and their complexes and to obtain highly purified vesicles morphologically corresponding to exosomes. Using affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we obtained extra-purified CD9+ and CD63+ exosomes, which simultaneously contain these two tetraspanins, while the CD81 tetraspanin was presented in a minor quantity. SDS-PAGE and MALDI analysis detected several major proteins with molecular masses over 10 kDa: CD9, CD63, CD81, lactadherin, actin, butyrophilin, lactoferrin, and xanthine dehydrogenase. Analysis of extracts by trifluoroacetic acid revealed dozens of peptides with molecular masses in the range of 0.8 to 8.5 kDa. Data on the uneven distribution of tetraspanins on the surface of horse milk exosomes and the presence of peptides open new questions about the biogenesis of these extracellular vesicles.

AB - Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we isolated highly purified vesicle preparations from 18 horse milk samples. Gel filtration of the initial preparations allowed us to remove co-isolating proteins and their complexes and to obtain highly purified vesicles morphologically corresponding to exosomes. Using affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we obtained extra-purified CD9+ and CD63+ exosomes, which simultaneously contain these two tetraspanins, while the CD81 tetraspanin was presented in a minor quantity. SDS-PAGE and MALDI analysis detected several major proteins with molecular masses over 10 kDa: CD9, CD63, CD81, lactadherin, actin, butyrophilin, lactoferrin, and xanthine dehydrogenase. Analysis of extracts by trifluoroacetic acid revealed dozens of peptides with molecular masses in the range of 0.8 to 8.5 kDa. Data on the uneven distribution of tetraspanins on the surface of horse milk exosomes and the presence of peptides open new questions about the biogenesis of these extracellular vesicles.

KW - Horses

KW - Animals

KW - Exosomes/metabolism

KW - Milk

KW - Proteins/metabolism

KW - Tetraspanins/metabolism

KW - Peptides/metabolism

KW - Chromatography, Affinity

KW - CD63

KW - extracellular vesicles

KW - peptides

KW - tetraspanins

KW - exosomes

KW - proteins

KW - CD9

KW - mass spectrometry

KW - horse milk

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85144637362&origin=inward&txGid=0b2b1c3e6b5df3345e145d27aa6dd1c2

UR - https://www.mendeley.com/catalogue/053343be-3ee9-3032-baf5-d9e033ecac80/

U2 - 10.3390/ijms232416106

DO - 10.3390/ijms232416106

M3 - Article

C2 - 36555744

VL - 23

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 24

M1 - 16106

ER -