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Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages

Published: June 30th, 2023



1School of Basic Medical Sciences, Anhui Medical University, 2State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, 3School of Life Sciences, Hebei University

This protocol describes a procedure to isolate small extracellular vesicles from macrophages by differential ultracentrifugation and extract the peptidome for identification by mass spectrometry.

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of <200 nm are present in various body fluids. These sEVs regulate various biological processes such as gene transcription and translation, cell proliferation and survival, immunity and inflammation through their cargos, such as proteins, DNA, RNA, and metabolites. Currently, various techniques have been developed for sEVs isolation. Among them, the ultracentrifugation-based method is considered the gold standard and is widely used for sEVs isolation. The peptides are naturally biomacromolecules with less than 50 amino acids in length. These peptides participate in a variety of biological processes with biological activity, such as hormones, neurotransmitters, and cell growth factors. The peptidome is intended to systematically analyze endogenous peptides in specific biological samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we introduced a protocol to isolate sEVs by differential ultracentrifugation and extracted peptidome for identification by LC-MS/MS. This method identified hundreds of sEVs-derived peptides from bone marrow-derived macrophages.

Small extracellular vesicles (sEVs) with a diameter of less than 200 nm are present in almost all types of body fluids and secreted by all kinds of cells, including urine, sweat, tears, cerebrospinal fluid, and amniotic fluid1. Initially, sEVs were considered as receptacles for disposing of cellular waste, which led to minimal research in the subsequent decade2. Recently, increasing evidence indicates that sEVs contain specific proteins, lipids, nucleic acids, and other metabolites. These molecules are transported to target cells3, contributing to intercellular communication, through which they pa....

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1. Isolation of small extracellular vesicles

NOTE: Perform all centrifugation in steps 1.1-1.11 at 4 °C.

  1. Preparation of sEVs-free fetal bovine serum (FBS): Centrifuge FBS overnight at 110,000 × g at 4 °C through an ultracentrifuge (see Table of Materials) to remove endogenous sEVs. Collect the supernatant, filter sterilize it with a 0.2 µm ultrafiltration membrane, and store it at -20 °C.
  2. Plate about 3 x .......

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For the sEVs isolated by differential ultracentrifugation (Figure 1), we evaluated their morphology, particle size distribution, and protein markers according to the International Society for Extracellular Vesicles (ISEV)17.

First, the morphology of sEVs was observed by TEM, showing a typical cup-like structure (Figure 2A). NTA showed that isolated sEVs were mostly concentrated at 136 nm (F.......

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When investigating the function of sEVs, it is imperative to attain high-purity sEVs from complex biological samples to avoid any potential contaminations. A variety of methods for sEVs isolation have been developed13, and among these methods, differential ultracentrifugation-based methods have shown relatively high purity of sEVs. In this study, 200 mL of cell supernatant was collected for 6 h, and about 200-300 µg of sEVs were obtained by differential ultracentrifugation. However, it should.......

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This study was supported by grants from the Natural Science Foundation of China (3157270). We thank Dr. Feng Shao (National Institute of Biological Sciences, China) for providing iBMDM.


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NameCompanyCatalog NumberComments
BCA Protein Assay KitBeyotime TechnologyP0012
CD9Beyotime TechnologyAF1192
Centrifugal filter tubeMilliporeUFC5010BK
Centrifuge bottles polypropyleneBeckman Coulter357003High-speed centrifuge
Chemiluminescent substrateThermo Fisher Scientific34580
DithiothreitolSolarbioD8220100 g
DMEM culture mediumCell WorldN?A
GRP94Cell Signaling Technology20292
High-speed centrifugeBeckman CoulterAvanti JXN-26Centrifuge rotor (JA-25.50)
Immortalized bone marrow-derived macrophages (iBMDM)National Institute of Biological Sciences, ChinaProvided by Dr. Feng Shao (National Institute of Biological Sciences, China)
IodoacetamideSigmal11495 g
Microfuge tube polypropyleneBeckman Coulter3574481.5 mL, Tabletop ultracentrifuge 
nano-high-performance LC systemThermo Fisher ScientificEASY-nLC 1000
Nanoparticle tracking analysis Malvern PanalyticalNanoSight LM10NanoSight NTA3.4
Orbitrap Q Exactive HF-X mass spectrometerThermo Fisher ScientificN/A
Phosphate-buffered salineSolarbioP1020
Polyallomer centrifuge tubesBeckman Coulter326823Ultracentrifuge
Protease inhibitorBimakeB14002
SpeedVac vacuum concentratorEppendorfConcentrator plus
Tabletop ultracentrifugeBeckman CoulterOptima MAX-XPUltracentrifuge rotor (TLA 55)
Transmission electron microscopeHITACHIH-7650B
UltracentrifugeBeckman CoulterOptima XPN-100Ultracentrifuge rotor (SW32 Ti)
Ultrasonic cell disruptorScientzSCIENTZ-IID
Western Blot imagerBio-RadChemiDocXRsImage lab 4.0 (beta 7)

  1. Kalluri, R., LeBleu, V. S. The biology, function, and biomedical applications of exosomes. Science. 367 (6478), (2020).
  2. Thery, C. Exosomes: secreted vesicles and intercellular communications. F1000 Biology Reports. 3, 15 (2011).
  3. Mathieu, M., Martin-Jaular, L., Lavieu, G., Thery, C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nature Cell Biology. 21 (1), 9-17 (2019).
  4. Chen, G., et al. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature. 560 (7718), 382-386 (2018).
  5. Ti, D., et al. LPS-preconditioned mesenchymal stromal cells modify macrophage polarization for resolution of chronic inflammation via exosome-shuttled let-7b. Journal of Translational Medicine. 13, 308 (2015).
  6. Sun, H., et al. Exosomal S100A4 derived from highly metastatic hepatocellular carcinoma cells promotes metastasis by activating STAT3. Signal Transduction and Targeted Therapy. 6 (1), 187 (2021).
  7. Xun, J., et al. Cancer-derived exosomal miR-138-5p modulates polarization of tumor-associated macrophages through inhibition of KDM6B. Theranostics. 11 (14), 6847-6859 (2021).
  8. Tai, Y. L., Chen, K. C., Hsieh, J. T., Shen, T. L. Exosomes in cancer development and clinical applications. Cancer Science. 109 (8), 2364-2374 (2018).
  9. Mashouri, L., et al. Exosomes: composition, biogenesis, and mechanisms in cancer metastasis and drug resistance. Molecular Cancer. 18 (1), 75 (2019).
  10. Yang, D., et al. Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics. Theranostics. 10 (8), 3684-3707 (2020).
  11. Zhang, Y., et al. Exosome: A review of its classification, isolation techniques, storage, diagnostic and targeted therapy applications. International Journal of Nanomedicine. 15, 6917-6934 (2020).
  12. Xu, R., Greening, D. W., Zhu, H. J., Takahashi, N., Simpson, R. J. Extracellular vesicle isolation and characterization: toward clinical application. The Journal of Clinical Investigation. 126 (4), 1152-1162 (2016).
  13. Li, P., Kaslan, M., Lee, S. H., Yao, J., Gao, Z. Progress in exosome isolation techniques. Theranostics. 7 (3), 789-804 (2017).
  14. Palanski, B. A., et al. An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease. Nature Communications. 13, 888 (2022).
  15. Kalaora, S., et al. Identification of bacteria-derived HLA-bound peptides in melanoma. Nature. 592 (7852), 138-143 (2021).
  16. Hamley, I. W. Small bioactive peptides for biomaterials design and therapeutics. Chemical Reviews. 117 (24), 14015-14041 (2017).
  17. Lotvall, J., et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. Journal of Extracellular Vesicles. 3, 26913 (2014).
  18. Thery, C., et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).
  19. Kim, Y. G., Lone, A. M., Saghatelian, A. Analysis of the proteolysis of bioactive peptides using a peptidomics approach. Nature Protocols. 8 (9), 1730-1742 (2013).
  20. Lyapina, I., Ivanov, V., Fesenko, I. Peptidome: Chaos or inevitability. International Journal of Molecular Sciences. 22 (23), 13128 (2021).
  21. Keller, M. D., et al. Decoy exosomes provide protection against bacterial toxins. Nature. 579 (7798), 260-264 (2020).
  22. Koeppen, K., et al. Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa. Proceedings of the National Academy of Sciences of the United States of America. 118 (28), e2105370118 (2021).

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