Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages
Summary
This protocol describes a procedure to isolate small extracellular vesicles from macrophages by differential ultracentrifugation and extract the peptidome for identification by mass spectrometry.
Abstract
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.
Introduction
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 participate in various biological processes, such as tissue repair, angiogenesis, immunity4 and inflammation5,6, tumor development and metastasis7,8,9, etc.
To facilitate the study of sEVs, it is imperative to isolate sEVs from complex samples. Different sEVs isolation methods have been developed based on the physical and chemical properties of sEVs, such as their density, particle size, and surface marker proteins. These techniques include ultracentrifugation-based methods, particle size-based methods, immunoaffinity capture-based methods, sEVs precipitation-based methods, and microfluidics-based methods10,11,12. Among these techniques, the ultracentrifugation-based method is widely recognized as the gold standard for sEVs isolation and is the most commonly used technique13.
An increasing amount of evidence suggests the presence of a multitude of undiscovered biologically active peptides in the peptidomes of various organisms. These peptides significantly contribute to numerous physiological processes by regulating growth, development, stress response14,15, and signal transduction16. The objective of sEVs' peptidome is to uncover the peptides carried by these sEVs and provide clues to their biological functions. Here, we present a protocol of isolating sEVs through differential ultracentrifugation, followed by extraction of peptides from these sEVs for further analysis of their peptidome.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| BCA Protein Assay Kit | Beyotime Technology | P0012 | |
| CD9 | Beyotime Technology | AF1192 | |
| Centrifugal filter tube | Millipore | UFC5010BK | |
| Centrifuge bottles polypropylene | Beckman Coulter | 357003 | High-speed centrifuge |
| Chemiluminescent substrate | Thermo Fisher Scientific | 34580 | |
| Dithiothreitol | Solarbio | D8220 | 100 g |
| DMEM culture medium | Cell World | N?A | |
| GRP94 | Cell Signaling Technology | 20292 | |
| High-speed centrifuge | Beckman Coulter | Avanti JXN-26 | Centrifuge rotor (JA-25.50) |
| Immortalized bone marrow-derived macrophages (iBMDM) | National Institute of Biological Sciences, China | Provided by Dr. Feng Shao (National Institute of Biological Sciences, China) | |
| Iodoacetamide | Sigma | l1149 | 5 g |
| Microfuge tube polypropylene | Beckman Coulter | 357448 | 1.5 mL, Tabletop ultracentrifuge |
| nano-high-performance LC system | Thermo Fisher Scientific | EASY-nLC 1000 | |
| Nanoparticle tracking analysis | Malvern Panalytical | NanoSight LM10 | NanoSight NTA3.4 |
| Orbitrap Q Exactive HF-X mass spectrometer | Thermo Fisher Scientific | N/A | |
| Phosphate-buffered saline | Solarbio | P1020 | |
| Polyallomer centrifuge tubes | Beckman Coulter | 326823 | Ultracentrifuge |
| Protease inhibitor | Bimake | B14002 | |
| SpeedVac vacuum concentrator | Eppendorf | Concentrator plus | |
| Tabletop ultracentrifuge | Beckman Coulter | Optima MAX-XP | Ultracentrifuge rotor (TLA 55) |
| Transmission electron microscope | HITACHI | H-7650B | |
| TSG101 | Sigma | AF8258 | |
| Ultracentrifuge | Beckman Coulter | Optima XPN-100 | Ultracentrifuge rotor (SW32 Ti) |
| Ultrasonic cell disruptor | Scientz | SCIENTZ-IID | |
| Western Blot imager | Bio-Rad | ChemiDocXRs | Image lab 4.0 (beta 7) |
| β-actin | Sigma | A3853 |
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