色谱2025，Vol.43Issue(5)：518-528,11.DOI:10.3724/SP.J.1123.2024.10014
脑脊液外泌体亚群的分离及其蛋白质组学分析
Isolation and proteomics analysis of cerebrospinal fluid exosome subtypes
摘要
Abstract
Exosomes are small extracellular vesicles 30-200 nm in diameter that contain many bioactive macromolecules,including proteins,lipids,and nucleic acids;consequently,they play important roles in many physiological and pathological processes and are classified into va-rious property-dependent subtypes.Research into exosome heterogeneity helps broaden our un-derstanding of the physiological and pathological mechanisms associated with exosomes.Exo-somes exist in many human biological fluids,with those derived from cerebrospinal fluid(CSF)regarded as potential disease biomarkers.Despite this,few studies have focused on their proteomics,and little research into CSF-derived exosome subtypes has been reported.Traumatic brain injury(TBI)is a major public health issue characterized by a large number of patients and complex pathological processes.While a comprehensive understanding of the pathophysiological processes that underpin TBI is essential for developing therapeutic interven-tions,proteomic studies into CSF-derived exosomes in patients with TBI are limited.Herein,we designed a tandem size-exclusion chromatography protocol for isolating and profiling the proteins of CSF-derived exosome subtypes from patients with TBI using nanoscale liquid chro-matography and trapped-ion mobility spectrometry time-of-flight mass spectrometry(nanoLC-TIMS-TOF-MS).We first centrifuged the collected CSF to remove cells and cell debris,after which it was concentrated by ultrafiltration to increase the exosome concentration and remove small proteins and peptides.A mini-size exclusion chromatography(Mini-SEC)column was then used to separate the exosomes from large amounts of interfering proteins,after which high performance liquid-SEC(HPL-SEC)was used to further separate exosomes according to size.The entire extracellular-vesicle-subset separation and purification process takes approximately 1 h for a single CSF sample.Four differently sized exosome subtypes were successfully isolated and are referred to as S1,S2,S3,and S4 in order of descending size.The S1 subtype exhibited the highest exosome purity according to the particle-to-protein ratio.Multiple characterization methods,including transmission electron microscopy(TEM),Western blotting(WB),and nanoparticle tracking analysis(NTA),confirmed that the exosome subtypes had been success-fully acquired.NanoLC-TIMS-TOF-MS,combined with database searching were then used to characterize the proteins.A total of 739 proteins were identified,of which 79%and 72%matched all proteins and the top 100 proteins in the Vesiclepedia database,respectively.Moreo-ver,gene ontology analysis revealed that the identified proteins are mainly located in extracellu-lar exosomes,and that the isolated exosome subtypes are closely related to multiple biological processes,including cell signaling,coagulation,and immune responses.Hierarchical cluster analysis revealed that samples from the same exosome subset are grouped first.Principal-com-ponent and Pearson's correlation coefficient analyses revealed that the proteins expressed in the CSF-derived exosome subtypes are heterogeneous.Interestingly,the proteins identified in the S1 subtype varied greatly between samples,highlighting the potential applicability of this subtype to formulating precise therapeutic regimens for different patients.We also analyzed the highly expressed proteins in the exosome subtypes,which revealed that the enrichment path-way of the S1 subtype involves Vitamin B12 metabolism and the regulation of protein catabolic processes,while the specific enrichment pathway of the S2 subtype includes binding and ligand uptake by scavenger receptors,heme scavenging from plasma,and an inflammatory response.In contrast,the unique enrichment pathway of the S3 subtype contains complementary and co-agulation cascades and acute-phase responses,while that of the S4 subtype includes post-trans-lational protein phosphorylation.Furthermore,STRING-based protein-association analysis pre-dicted multiple interactions among proteins in the various exosome subtypes.In conclusion,the developed tandem size-exclusion chromatography method was used to isolate cerebrospinal flu-id exosome subtypes.This study enriches knowledge regarding cerebrospinal fluid exosomes in patients with TBI based on proteomics.关键词
外泌体/外泌体亚群/蛋白质组学/脑脊液/创伤性脑损伤Key words
exosomes/exosome subtypes/proteomics/cerebrospinal fluid(CSF)/traumatic brain injury(TBI)分类
化学化工引用本文复制引用
陈晓菲,刘威,张文镓,李言鹏,王志华,高明霞,张祥民..脑脊液外泌体亚群的分离及其蛋白质组学分析[J].色谱,2025,43(5):518-528,11.基金项目
国家自然科学基金(22274027) （22274027）
上海市浦东新区卫生计生科研项目(PW2021A-70).National Natural Science Foundation of China(No.22274027) （PW2021A-70）
Health Science and Technology of the Pud-ong Municipal Commission of Health Committee of Shanghai(No.PW2021A-70). （No.PW2021A-70）
