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Int J Mol Sci. 2019 May 16;20(10). pii: E2434. doi: 10.3390/ijms20102434.

Extra Purified Exosomes from Human Placenta Contain An Unpredictable Small Number of Different Major Proteins.

Author information

1
SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., 630090 Novosibirsk, Russia. jorochka24@mail.ru.
2
SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., 630090 Novosibirsk, Russia. feabelit@mail.ru.
3
Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of Russian Academy of Sciences, 690022 Vladivostok, Russia. bulgakov-dv@mail.ru.
4
G. B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS, 159 100 let Vladivostoku Ave., 690022 Vladivostok, Russia. paveldmt@piboc.dvo.ru.
5
SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., 630090 Novosibirsk, Russia. valentin.vlassov@niboch.nsc.ru.
6
SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., 630090 Novosibirsk, Russia. lenryab@yandex.ru.
7
SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., 630090 Novosibirsk, Russia. sirozha@gmail.com.
8
SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., 630090 Novosibirsk, Russia. nevinsky@niboch.nsc.ru.

Abstract

Exosomes are nanovesicles (30-100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30-100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins' tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11-13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes.

KEYWORDS:

exosomes purification; human placenta; proteins identification; proteins separation

PMID:
31100946
DOI:
10.3390/ijms20102434
Free PMC Article

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