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 pa....

Protocol

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 10⁷ immortalized bone marrow-derived macrophages (iBMDMs) on a 150 mm culture dish and add 20 mL of DMEM culture medium (see Table ....

Results

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.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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