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Chinese Journal of Chromatography
2025, Vol. 43, No. 5
Online: 08 May 2025

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Preface for Special Issue of Separation and Analysis for Exosome HU Lianghai, QIAO Xiaoqiang, TAO Weiguo 2025, 43 (5):  397-398.  DOI: 10.3724/SP.J.1123.2025.01023 Abstract ( 58 )   HTML ( 20 )   PDF (788KB) ( 49 )

Perspectives

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Progress and prospect of separation and analysis of single-cell and single-particle exosomes BU Aixiang, WU Guangyao, HU Lianghai 2025, 43 (5):  399-412.  DOI: 10.3724/SP.J.1123.2024.11001 Abstract ( 97 )   HTML ( 15 )   PDF (2353KB) ( 38 )   Exosomes are nanoscale vesicles secreted by cells and are encapsulated in lipid bilayers. They play crucial roles in cell communication and are involved in a variety of physiological and pathological processes, including immune regulation, angiogenesis, and tumor initiation and metastasis. Exosomes carry a variety of biomolecules from maternal cells and are therefore important vehicles for discovering disease markers. Traditional detection methods only provide average cell-population information for a given sample and cannot establish clear relationships between the biological functions of exosomes and subtype owing to the significant heterogeneity associated with exosomes from different cell subsets. Therefore, characterizing exosomes at the single-cell and single-particle levels requires exosome specificities to be further explored and the characteristics of various exosome subtypes to be distinguished. Commonly used single-particle exosome characterization technologies include flow cytometry, super-resolution microscopy, atomic force microscopy, surface-enhanced Raman spectroscopy, proximity barcoding assay and MS. In this paper, we summarize recent advances in the separation and characterization of single-cell exosomes based on microfluidics and provide future applications prospects for emerging technologies (such as Olink proteomics, click chemistry, and molecular imprinting) for studying single-cell and single-particle exosomes.

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Typical strategy and research progress of efficient isolation methods of exosomes based on affinity interaction WANG Haiyan, XIE Peijuan, QIAO Xiaoqiang, ZHANG Liyuan 2025, 43 (5):  413-423.  DOI: 10.3724/SP.J.1123.2024.11004 Abstract ( 127 )   HTML ( 14 )   PDF (7267KB) ( 57 )   Exosomes form a subclass of extracellular vesicle that are secreted by most cells and found in nearly all body fluids, including blood, urine, saliva, amniotic fluid, and milk, as well as in various tissues and intercellular spaces. Exosomes have recently been recognized as crucial intercellular communication mediators, and an increasing number of studies have shown that exosomes are important liquid-biopsy tools that play irreplaceable roles in the diagnosis, prognosis, and treatment of diseases. The ability to isolate high-quality exosomes is a prerequisite for diagnosing and subsequently treating diseases in an accurate and repeatable manner. However, efficiently isolating exosomes from complex biological samples is challenging owing to their relatively low abundances and interference from non-vesicular macromolecules (such as cell debris and proteins). To date, various isolation techniques based on the physical, chemical, and biological characteristics of exosomes have been developed. Indeed, efficient affinity-interaction-based methods have recently overcome the limitations and drawbacks of traditional exosome isolation methods and are widely used in scientific research and clinical applications. This review focuses on exosome isolation and enrichment, and systematically reviews recent research progress on efficient isolation methods based on affinity interactions. Developmental prospects of exosome isolation and enrichment directions are analyzed with the aim of providing a reference for the construction and use of new exosome-isolation strategies.

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Advances in exosome-targeting aptamer-screening techniques ZHENG Liting, YANG Ge, QU Feng 2025, 43 (5):  424-433.  DOI: 10.3724/SP.J.1123.2024.10029 Abstract ( 79 )   HTML ( 6 )   PDF (1579KB) ( 21 )   Exosomes play crucial intercellular-communication roles and regulate various cellular physiological processes. They are considered potential biomarkers for the early diagnosis of cancers and other diseases. Therefore, detecting and isolating exosomes with specific functions has significant clinical implications. Moreover, the development of low-cost, highly sensitive recognition elements for identifying exosomes is essential for advancing early disease diagnosis and treatment. Nucleic acid aptamers are single-stranded DNA or RNA molecules capable of specifically binding to targets and are produced through the systematic evolution of ligands by exponential enrichment (SELEX) technique. Such aptamers are highly stable, chemically synthesizable, exhibit high affinities and specificities, and are applicable to a broad range of targets, which endow them with unique advantages. Currently, aptamers that target exosomes have been used in a variety of research fields, including cell imaging, drug delivery, and disease diagnosis and treatment. However, selecting aptamers that precisely identify specific exosomes is significantly challenging owing to the complex structures of exosome and their heterogeneity. Consequently, obtaining high-performance aptamers requires efficient screening techniques. This review first summarizes the functions and selection strategies of key targets for exosome-aptamer screening. Furthermore, it outlines the main methods and techniques currently used to screen exosome aptamers, which includes five screening techniques: magnetic bead-SELEX, microfluidic-SELEX, nitrocellulose-SELEX, cell-SELEX, and capillary electrophoresis-SELEX. The separation principles, advantages, limitations, and the latest applications of these techniques are discussed in detail. The review finally addresses current challenges associated with selecting exosome aptamers and provides insight into future research directions.

Reviews

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Exosome separation and enrichment technologies and their applications in disease diagnosis and treatment HOU Guoshan, YUAN Huiming, LIANG Zhen, ZHANG Lihua, ZHANG Yukui 2025, 43 (5):  434-445.  DOI: 10.3724/SP.J.1123.2024.09007 Abstract ( 104 )   HTML ( 11 )   PDF (2076KB) ( 90 )   Exosomes are nanoscale vesicles wrapped in lipid bilayers that are secreted by cells and carry a variety of proteins, lipids, nucleic acids, and metabolites. Exosomes are widely present in various bodily fluids and mediate intercellular communication. They participate in a variety of physiological and pathological processes, including immune regulation, angiogenesis, tumorigenesis, and metastasis, and have significant clinical diagnosis and treatment potential. Exosomes are source-rich, structurally stable, and reflect the states of their parental cells. Therefore, they are expected to serve as novel diagnostic markers for various diseases. In addition, stem-cell-derived exosomes show therapeutic potential and have the advantages of low immunogenicity, high safety and easy storage, and exhibit therapeutic potential for neurodegenerative disorder, cardiovascular disease, and cancer. Furthermore, exosomes are highly biocompatible, have natural homing properties, and are capable of easily penetrating biological barriers, making them excellent drug-delivery carriers. Isolation and enrichment of exosomes is a prerequisite for downstream analysis and application. High-purity, high-yield, and high-throughput exosome-isolation methods are expected to be used in clinical diagnosis and treatment applications. Based on the physicochemical properties of exosomes, including density, size, charge, and surface composition, exosome-isolation methods are mainly divided into density-based (e.g., differential ultracentrifugation, density-gradient ultracentrifugation), size-based (e.g., ultrafiltration, size-exclusion chromatography, field-flow fractionation), polymer-precipitation (e.g., polyethylene-glycol-based precipitation), and chemical affinity (e.g., antibody-based, aptamer-based, and surface-lipid-based lipid probes) methods. Currently, basic research into exosomes and their clinical applications face a number of challenges. Firstly, the complexity and heterogeneity of exosomes and the lack of standardized isolation methods has led to highly variable research results that hinder comparing and reproducing results between different laboratories and clinical settings. Current isolation methods are generally hindered by insufficient purity, low yield, low throughput, and difficulties separating specific subpopulations, which seriously restrict the development of the exosome field. Secondly, exosome-isolation methods that are easy to use in the clinic, have few technical requirements, and are highly efficient and inexpensive are lacking. Commonly used classical methods, such as ultracentrifugation, are time-consuming, labor-intensive, require large sample volumes, and are inappropriate for clinical settings. Methods such as immunoaffinity can be used to isolate exosomes from precious trace samples in clinical practice; however, high costs, low recoveries, and high operating requirements are shortcomings that restrict sample analysis in the clinic. In addition, robust large-scale methods for preparing exosomes are lacking. There is an urgent need to develop repeatable and scalable methods for preparing batches of high-quality exosomes owing to the rapid development of exosomes for the treatment of clinical diseases. Generally, exosome research progress is expected to greatly improve our understanding of the biological functions and components of exosomes, which will help transform the exosome research into effective diagnostic and therapeutic strategies and lead to new precision-medicine and personalized-treatment applications. This article summarizes the latest progress in exosome-isolation and -enrichment technologies and introduces the application of exosomes as disease diagnostic markers, therapeutic agents, and drug delivery carriers. Finally, the future developmental trends in exosome isolation and enrichment technologies for disease diagnosis and treatment are discussed.

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Research progress of peptide recognition-guided strategies for exosome isolation and enrichment XU Kun, HUANG Yanyan, ZHAO Rui 2025, 43 (5):  446-454.  DOI: 10.3724/SP.J.1123.2024.10015 Abstract ( 85 )   HTML ( 3 )   PDF (1970KB) ( 43 )   Exosomes are bilayered vesicles derived from living cells and bacteria that are loaded with abundant biomolecules, such as proteins and nucleic acids. As an important medium of remote cell communication, exosomes are closely related to the occurrence and development of a number of diseases, including those involving tumors and inflammation. The isolation and enrichment of exosomes in complex biosystems is greatly significant for the diagnosis, prognosis, and detection of diseases, as well as in molecular-mechanism research. However, exosomes are usually nanoscale size distribution and widely existed in complex biological samples, including blood, tissue fluids, and urine, which bring difficulties and challenges to the isolation and enrichment of exosomes. To address this issue, several methods based on the physical properties of exosomes have been developed. For example, exosomes can be obtained by ultracentrifugation at high centrifugal force based on density differences; they can also be isolated and enriched by size-exclusion chromatography and ultrafiltration based on size heterogeneity. Exosomes can also be separated in high yields but with low purities using commercial polymer-coprecipitation-based isolation kits. While the abovementioned methods can be used to isolate and enrich exosomes in a highly efficient manner, accurately distinguishing interfering particles, including protein aggregates and microvesicles, in biosystems is still difficult, resulting in the poor purity of exosome isolation and enrichment. Affinity ligands are widely used during the affinity isolation and enrichment of exosomes. Antibodies exhibit high selectivity and affinity; consequently exosomes can be captured highly selectively by exploiting specific antigen/antibody interactions. However, antibodies also have some limitations, including complex preparation procedures, high costs, and poor stability. Chemical affinity ligands, such as aptamers, peptides, and small molecules, are also widely used to isolate and enrich exosomes. As a kind of molecular recognition tool, peptides contain a variety of amino acids and exhibit many advantages, including good biocompatibility, low immunogenicity, and design flexibility. Solid-phase synthesis strategies have rapidly developed, thereby providing a basis for automated and large-scale peptide synthesis. Affinity peptides have been widely used to recognize and analyze target biomolecules in complex physiological environments in a highly selective manner. A series of protein-targeting peptides has been reported based on the biomolecular characteristics of exosomes. These affinity peptides can be specifically anchored onto highly enriched transmembrane proteins on exosome surfaces, thereby enabling the efficient and highly selective isolation and enrichment of exosomes in complex systems. Additionally, exosomes contain stable bilayer membranes consisting of abundant and diverse phospholipid molecules. The development of phospholipid-molecule-targeting peptides is expected to effectively eliminate interference from protein aggregates and other particles. In addition to differences in the compositions of phospholipids in biofilms, exosomes are smaller and more curved than apoptotic bodies and microvesicles. A series of affinity peptides capable of inducing and sensing high membrane curvatures are widely used to isolate and enrich exosomes. The tumor microenvironment can produce and release tumor-derived exosomes that are buried in a large number of normal cell-derived exosomes. Accordingly, pH-responsive peptides have been designed and modified based on the acidic environments of tumor-derived exosomes, which were accurately and tightly anchored via peptide insertion and folding. Focusing on the current status of exosome research, this paper introduces and summarizes current and widely used methods for isolating and enriching exosomes. Various exosome-targeting peptide-design and screening principles are introduced based on the characteristics and advantages of peptides. The applications of these peptides to the isolation and enrichment of exosomes are also summarized, thereby providing strong guidance for the efficient and highly selective isolation and enrichment of exosomes in complex life-related systems.

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Exosome separation and analysis based on microfluidics technology and its clinical applications XING Yuhang, REN Xiangshan, LI Donghao, LIU Lu 2025, 43 (5):  455-471.  DOI: 10.3724/SP.J.1123.2024.10032 Abstract ( 44 )   HTML ( 3 )   PDF (3766KB) ( 18 )   Exosomes are cell-secreted nanoscale vesicles 30-150 nm in size and encompass a diverse array of biomolecules, including lipids, proteins, and nucleic acids. Exosomes play pivotal roles during the intercellular exchange of materials and information, and are closely associated with the onset and progression of a variety of diseases. Therefore, comprehensively investigating exosomes is very important in terms of disease diagnosis and treatment. However, exosomes are genetically heterogeneous and are composed of different materials. Additionally, exosome-size and packing-specific-biomarker heterogeneities result in biofunction diversity. Moreover, isolating and analyzing exosomes is highly challenging owing to their small sizes and heterogeneities. Accordingly, effective separation methods and analytical techniques for highly specifically and efficiently identifying exosomes are urgently needed in order to better understand their functionalities. While separation and analysis is required to reveal exosome heterogeneity, the former is confronted by three primary challenges. Firstly, exosome heterogeneity (including heterogeneous marker expressions and size heterogeneity that results in heterogeneous functions) results in systems that are very difficult to separate. Secondly, the coexistence of non-vesicular contaminants (lipoprotein nanoparticles, soluble proteins, nucleic acids, etc.) and the complex matrix effects of body fluids also contribute to separation difficulties. Thirdly, enrichment is a highly challenging task owing to low exosome concentrations. Traditional methods, such as ultracentrifugation and size-exclusion chromatography, fall short in terms of their abilities to precisely separate and analyze exosomes. On the other hand, microfluidics has emerged as a robust tool for the efficient analysis of complex biological samples and is characterized by miniaturization, precise control, high throughput, automation, and integration. Firstly, the operability, integrability, and modifiability of a microfluidics system facilitate exosome separation and purification based on surface properties, size, charge, and polarity. Secondly, the use of a microfluidics approach, with its high throughput, low reagent consumption, and multichannel manipulability, greatly facilitates preparing exosomes and enhancing their concentrations. Thirdly, microfluidics ensures that diverse separation methods are compatible with downstream analysis techniques. Exosomes are highly heterogeneous; hence, they are classified by type and subpopulation (according to origin, size, molecular markers, functions, etc.). This paper first discusses microfluidics techniques for separating exosomes and examines various separation strategies grounded in the physicochemical properties of exosomes. We then analyze exosome detection methodologies that use microfluidics platforms and encompass traditional group-exosome analysis techniques and novel single-exosome analysis approaches. Finally, we discuss future clinical applications of microfluidics technology in exosome research, particularly its potential for diagnosing and treating diseases, thereby underscoring the applications value of microfluidics technology in the realm of personalized and precision medicine. Furthermore, cutting-edge microfluidics platforms offer novel perspectives for purifying and preparing EVs owing to precise fluid control, integration, miniaturization, and high-throughput characterization. EV populations, subpopulations, and single vesicles can be purified based on their physicochemical properties and microfluidics features. Comprehensive lab-on-a-chip methods are promising in terms of separating EVs based on traits, such as size, surface markers, and charge, and for obtaining highly pure EVs. Recycled EV samples can be prepared by controlling the high-throughput and multichannel capabilities of microfluidics approaches. The transition from bulk EV analysis to single-vesicle analysis provides opportunities to explore the heterogeneous nature of EVs, thereby augmenting their potential for disease diagnosis.

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Research advances of liposomes and exosomes in drug delivery and biomarker screening SU Yating, QIAN Xiaohong, QIN Weijie 2025, 43 (5):  472-486.  DOI: 10.3724/SP.J.1123.2024.08012 Abstract ( 92 )   HTML ( 3 )   PDF (1683KB) ( 30 )   Vesicles, are categorized as artificial (i.e., liposomes) or natural (i.e., extracellular vesicles (EVs)) and play significant roles in drug-delivery and biomarker-screening applications. Liposomes, as a representative form of artificial vesicle, are spherical lipid structures composed of one or more artificially synthesized phospholipid bilayers. Liposomes are highly biocompatible and bioavailable, very stable, and easily synthesized; hence, they are among the most commonly used and frequently applied nanocarriers in targeted drug-delivery systems (DDS). EVs are natural small membrane-bound vesicles actively secreted by cells and contain a variety of components, including nucleic acids, proteins, and lipids. They also serve as important mediators of intercellular communication. As the smallest EV subtype, with diameters of only 30-100 nm, exosomes contain unique biomolecules that are considered to be the fingerprints of the parent cells. In the pathological state, the content of exosomes will change; consequently, exosomes are potential disease-diagnosis biomarkers. Recent clinical trials have shown that exosomes are ideal nanocarriers in targeted drug-delivery therapies for a variety of diseases. Compared with traditional artificial liposomal carriers, exosomes display unique advantages and provide the DDS field with new possibilities. Liposomes and exosomes are receiving increasing levels of attention in the drug-delivery and biomarker-screening fields. This article introduces techniques for the preparation of liposomes, and the enrichment and separation of exosomes, and delves into research progress on their use in drug-delivery and biomarker-screening applications. Finally, challenges facing the use of liposomes and exosomes in clinical applications are discussed.

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Recent advances on the role of exosomes in neurodegenerative diseases BU Caiting, ZHU Xuedong, ZHANG Qianying, SHAO Wenya 2025, 43 (5):  487-497.  DOI: 10.3724/SP.J.1123.2024.10035 Abstract ( 71 )   HTML ( 3 )   PDF (1564KB) ( 16 )   Exosomes are nano-sized, lipid bilayer vesicles secreted by cells. They carry essential bioactive molecules, such as proteins, nucleic acids, and lipids, and are widely present in bodily fluids including blood and cerebrospinal fluid. Exosomes transfer bioactive molecules to target cells through various mechanisms, including endocytosis, ligand-receptor interactions, or direct membrane fusion, and play crucial roles in intercellular communication, including facilitating intercellular information exchange, maintaining nerve-cell function, participating in immune responses, and providing nutritional support. Exosomes significantly promote signal transmission and intercellular communication in the central nervous system and are involved in the pathogenesis and development of diseases by participating in the spread of pathological proteins, regulating neuroinflammation, and the deposition of pathological proteins. Therefore, exosomes play key roles in the occurrence and development of neurodegenerative diseases, and their contents, especially proteins and miRNAs, are specific for given pathological and physiological states and are relatively stable during extraction and analysis. Hence, exosomes are ideal tools for diagnosing diseases, staging their courses, and assisting prognosis. This article further explores exosomes derived from blood, saliva, urine, and cerebrospinal fluid as potential diagnostic biomarkers for neurodegenerative diseases. As natural drug-delivery systems, exosomes have the advantages of biocompatibility, ability to cross biological barriers, target specificity, stability, and containing natural therapeutic molecules, which can effectively improve the precision and efficacy of drug delivery and reduce side effects, making them an ideal carrier for delivering drugs to the central nervous system. Therefore, exosomes hold great potential in the diagnosis and treatment of central nervous system diseases. This article systematically reviews the latest advances in exosome research directed toward specific neurodegenerative diseases, focusing on their roles played in disease pathogenesis, progression, diagnosis, and treatment, with the aim of providing theoretical support and a reference for the early diagnosis and treatment of these diseases.

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Bibliometric analysis of exosomes in the biomarker research field HUANG Yuxia, WANG Haiyan, ZHANG Yihan, LIN Yifei, QIAO Xiaoqiang, HU Lianghai 2025, 43 (5):  498-507.  DOI: 10.3724/SP.J.1123.2025.01025 Abstract ( 68 )   HTML ( 2 )   PDF (2871KB) ( 13 )   Exosomes are extracellular vesicles secreted by cells and are rich in genetic material and proteins. The surfaces of exosome membranes contain many blast-specific markers that provide an important basis for disease diagnosis, progression, and treatment. Herein, we searched the Web of Science core collection (SCI-EXPENED) database for research and review articles on “exosomes” and “biomarkers” or “diagnostics” or “liquid biopsy” as research topics between 2010 and 2024. Bibliometric analysis revealed that exosomes have received increasing levels of attention as disease biomarkers, with China contributing the most to these studies. Herein, we focus on marker diagnoses for cancer, inflammation, and diabetes, as well as neurodegenerative and cardiovascular diseases. Chromatography, mass spectrometry, Raman spectroscopy, and other techniques are typically used to analyze exosomal nucleic acids, proteins, and metabolites, with commonly used test samples including plasma, serum, urine, saliva, cerebrospinal fluid, and other bodily fluids. Research into exosomes as tumor markers has mainly focused on eight highly prevalent cancers, including lung, breast, and prostate cancers. This paper focuses on exosomes as disease biomarkers and uses a bibliometric tool system to analyze the use of exosomes and their contents as biomarkers in the disease diagnosis field between 2010 and 2024, analyzes development prospects, and discusses future exosome-mediated efforts for diagnosing and treating diseases, and is expected to provide a reference for studying and applying exosomes as disease markers.

Articles

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Efficient capture and proteomics analysis of urinary extracellular vesicles by affinity purification ZHANG Guiyuan, ZHAN Zhen, TAO Weiguo, ZHANG Hao 2025, 43 (5):  508-517.  DOI: 10.3724/SP.J.1123.2024.11013 Abstract ( 138 )   HTML ( 9 )   PDF (3915KB) ( 48 )   Liquid biopsy is a promising alternative to traditional tissue biopsies for diagnosing cancer because it offers advantages such as minimal invasiveness, accessibility, and ease of operation. Extracellular vesicles (EVs) are lipid bilayer vesicles that contain proteins, DNA, and RNA and are secreted by cells. Indeed, urinary EVs are important sources of cancer biomarkers. The lipid bilayer protects EV proteins from degradation by enzymes present in bodily fluids. Prostate cancer (PCa) is among the most prevalent malignancies in developed countries and is the second-leading cause of cancer-related mortality in men. Current screening methods commonly used to initially evaluate patients with suspected PCa include serum prostate-specific antigen (PSA) testing and digital rectal examination (DRE), with magnetic resonance imaging (MRI) and transrectal ultrasound often recommended for further assessment. However, both PSA testing and DRE have limited specificities, which results in a substantial number of unnecessary prostate biopsies. Consequently, additional reliable biomarkers need to be urgently discovered for rapidly diagnosing PCa more accurately. Prostate-derived secretions, including those associated with malignancies, are detectable in urine owing to the anatomical proximity of the prostate to the urethra; hence urine is a promising liquid-biopsy medium for discovering PCa biomarkers, which is a topic that has been the focus of extensive research efforts in recent years. However, isolating EVs from biofluids in sufficient yields for proteomics analysis remains challenging. In this study, functional magnetic beads EVlent (extracellular vesicles isoLated efficiently, naturally, and totally) with high-affinity capabilities were developed for selectively enriching EVs from biological fluids.The surfaces of the beads were modified with three antibodies that target CD9, CD63, and CD81, which enables the specific recognition of EV surface proteins. The isolation performance of EVlent was validated by comprehensively characterizing urinary EVs using Western blotting (WB), nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). WB revealed prominent bands for EV markers (CD9, TSG101, and HSP70) in EVlent-enriched samples, whereas weaker bands were observed following ultracentrifugation (UC). NTA revealed that the EVs isolated by EVlent are predominantly in the 50-400 nm size range, with a content of 4.1×109 particles/mL, which is significantly higher than the value of 1.8×109 particles/mL obtained by UC. TEM confirmed that the isolated EVs have characteristic elliptical or cup-shaped vesicular structures. These findings demonstrate that EVlent outperforms UC in terms of enrichment efficiency and purity, delivering a separation efficiency of 87.2% compared to the value of 30.3% obtained by UC. We used proteomics to analyze urinary EVs isolated from 15 healthy volunteers and 15 patients with prostate cancer using EVlent affinity magnetic beads with the aim of identifying potential biomarkers for prostate cancer. On average, 2039 proteins and 14490 peptides were identified in the control group, while 1982 proteins and 13100 peptides were identified in the patient group. Further analysis revealed 91 proteins commonly found in the Vesiclepedia database (Top 100). Compared with the healthy volunteers, 88 proteins were upregulated and 90 proteins were downregulated in patients with prostate cancer. Gene ontology (GO) analysis showed that these upregulated proteins are enriched in extracellular exosomes, extracellular space, extracellular region, collagen-containing extracellular matrix, proteolysis and protein-binding. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) highlighted associations between ribosomes, protein digestion and absorption, complement and coagulation cascades, prostate cancer, transcriptional misregulation in cancer, aldosterone-regulated sodium reabsorption, endocrine and other factor-regulated calcium reabsorption, and pancreatic secretion. Notably, four proteins including plasminogen activator urokinase (PLAU), platelet-derived growth factor subunit A (PDGFA), matrix metalloproteinase 3 (MMP3), and neuroblastoma RAS viral oncogene homolog (NRAS) were identified within the prostate cancer pathway, highlighting their potential as biomarkers for the early diagnosis and prognosis of prostate cancer. In conclusion, this study introduced EVlent as a robust platform for the efficient isolation and proteomics analysis of EVs, providing valuable insight into urinary EV biomarkers and their clinical prostate-cancer applications.

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Isolation and proteomics analysis of cerebrospinal fluid exosome subtypes CHEN Xiaofei, LIU Wei, ZHANG Wenjia, LI Yanpeng, WANG Zhihua, GAO Mingxia, ZHANG Xiangmin 2025, 43 (5):  518-528.  DOI: 10.3724/SP.J.1123.2024.10014 Abstract ( 120 )   HTML ( 10 )   PDF (3792KB) ( 42 )   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 various property-dependent subtypes. Research into exosome heterogeneity helps broaden our understanding of the physiological and pathological mechanisms associated with exosomes. Exosomes 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 interventions, 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 chromatography 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 successfully 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. Moreover, gene ontology analysis revealed that the identified proteins are mainly located in extracellular 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-component 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 pathway 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 coagulation cascades and acute-phase responses, while that of the S4 subtype includes post-translational protein phosphorylation. Furthermore, STRING-based protein-association analysis predicted multiple interactions among proteins in the various exosome subtypes. In conclusion, the developed tandem size-exclusion chromatography method was used to isolate cerebrospinal fluid exosome subtypes. This study enriches knowledge regarding cerebrospinal fluid exosomes in patients with TBI based on proteomics.

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Isolation and proteomic analysis of bacterial outer membrane vesicle subpopulations YU Poju, ZOU Xun, WU Yan, LI Suntao, XIAO Hua 2025, 43 (5):  529-538.  DOI: 10.3724/SP.J.1123.2024.10028 Abstract ( 200 )   HTML ( 10 )   PDF (3867KB) ( 107 )   Outer membrane vesicles (OMVs) are 20-400 nm in size, membrane-bound, and secreted by gram-negative bacteria. OMVs play important roles in processes such as toxin delivery and immune evasion. Although many studies have revealed the critical roles played by OMVs, their heterogeneity has limited our ability to attain a comprehensive understanding of their protein compositions and functions. Therefore, studying the compositions of heterogeneous OMVs subpopulations and their biological functions is important. Herein, we used ultracentrifugation combined with density-gradient centrifugation and quantitative proteomics to systematically separate, characterize, and comprehensively analyze OMVs secreted by Escherichia coli DH5α and Pseudomonas aeruginosa PAO1. First, crude OMVs extracts from both strains were obtained by ultracentrifugation and subjected to iodixanol density-gradient centrifugation to afford six fractions each. DH5α-OMVs and PAO1-OMVs particle-size distributions were then determined via nanoparticle tracking analysis, with average particle sizes of 131.0-161.0 and 140.0-169.0 nm determined for the two subpopulation, respectively. Vesicles were observed to have classical chattel structures by transmission electron microscopy. OMVs subpopulation distributions in the density-gradated fractions were determined by silver staining and protein immunoblotting, which also identified F1a-F4a and F1b-F5b as the effective DH5α-OMVs and PAO1-OMVs subpopulation fractions, respectively. We then identified 2388 and 905 proteins from the DH5α-OMVs and PAO1-OMVs subpopulation, respectively, and used k-means clustering and gene ontology (GO) enrichment analyses to reveal the heterogeneities of the various density subpopulations in terms of biological functions, such as energy metabolism, material transport and ribosome synthesis. Comparative analysis of the E. coli DH5α-OMVs and P. aeruginosa PAO1-OMVs subpopulations finally revealed that they exhibit different functional characteristics, despite sharing commonalities in their basic OMVs functions. The F1a DH5α-OMVs subpopulation was found to be enriched for functions related to amino-acid metabolism and protein synthesis, while the F2b PAO1-OMVs subpopulation exhibited significant biomolecule synthesis functions. This study revealed that bacterial OMVs subpopulations have distinct biological functions, which in turn provides a new theoretical basis for understanding the pathogenic mechanisms of bacteria and their interactions with the host, thereby expanding their biological applications.

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Enriching plasma exosomes for proteomic analysis using a phosphatidylserine-imprinted polymer CHENG Xianhui, YU Wenjing, WANG Dongxue, JIANG Liyan, HU Lianghai 2025, 43 (5):  539-546.  DOI: 10.3724/SP.J.1123.2024.05003 Abstract ( 123 )   HTML ( 18 )   PDF (2984KB) ( 45 )   Exosomes are 40-160 nm vesicular nano-bodies secreted by most cells that carry large amounts of biologically active substances originating from the parent cell. Proteins in exosomes are protected by phospholipid bilayer membranes that protect them from degradation by enzymes within body fluids. Along with nucleic acid, proteins and metabolites, exosomes are biomolecules that are considered to be among the most important for discovering tumor markers. Plasma is among the most commonly used body fluids in clinical settings; it is highly complex and contains many proteins and metabolites that interfere with exosome isolation. Consequently, the development of methods for effectively isolating exosomes is a key challenge prior to their use in clinical research. In this study, we used a phosphatidylserine molecularly imprinted polymer (PS-MIP) to enrich plasma exosomes. Subsequent immunoblotting analyses for the CD9, TSG101, and CD81 exosome marker proteins showed that signals can be detected using only 5 μL of plasma, thereby demonstrating the efficiency and specificity of the enrichment protocol. Transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA) data revealed that the enriched vesicles are 30-100 nm in size with elliptical or cup-shaped structures, consistent with the morphology and particle-size-distribution characteristics of the exosomes, suggesting that PS-MIP is capable of successfully isolating exosomes. Nanoflow cytometry revealed that 75.4% of the multi-angle laser scattering (MALS) signal is derived from the PS-MIP-enriched exosomes, which indicates that these enriched exosomes are highly pure and free of interference from impurities, such as aggregated protein particles that are similar in size to the exosomes themselves. This method was used to analyze the proteomes and potential exosomal protein markers of clinical plasma samples from three pancreatic-cancer patients and three healthy volunteers. A total of 1052 proteins and 4545 peptides were identified in the plasma exosomes of healthy volunteers, with a total of 972 proteins and 4096 peptides identified in the plasma exosomes of the pancreatic-cancer patients. Further bioinformatics analyses revealed that the Vesiclepedia database covered 84% of the proteins identified in the plasma exosomes isolated using the PS-MIP method; these proteins comprise 77 of the 100 most frequently identified exosomal proteins in the ExoCarta database. The identified proteins from the cellular components were subjected to gene ontology (GO) analysis, which revealed that they are mainly derived from the exosomes, thereby demonstrating the high selectivity of the PS-MIP method for enriching plasma exosomes and providing specificity for subsequent tumor-marker screening. Label-free quantitative analysis showed that 11 proteins were upregulated and 24 proteins were downregulated in the plasma exosomes of patients with pancreatic cancer compared to those of healthy volunteers. The highly expressed and lowly expressed proteins in the plasma exosomes of patients with pancreatic cancer were subjected to GO, which showed that highly expressed proteins related to the positive regulation of metabolic and biological processes were found in the plasma exosomes of patients with pancreatic cancer compared to those of healthy volunteers, whereas the most significantly under-expressed proteins are related immune-system processes, followed by stimulus-responsive, multicellular bioprocesses, bioregulatory, and interspecies-interacting biological-process-related proteins. The top three proteins, which are relatively highly correlated through protein-protein interaction networks (PPI) analysis, were determined to be complement factor D (CFD), complement component 3 (C3), and von Willebrand factor (VWF). Among the upregulated proteins in the exosomes of patients with pancreatic cancer, exostosin-like glycosyltransferase 2 (EXTL2), α-2-macroglobulin like 1 (A2ML1), and Parkinson’s disease protein 7 (PARK7) were the most significantly overexpressed. Hence, these proteins are potential biomarkers for the diagnostic and prognostic assessment of pancreatic cancer and may provide support for further clinical studies into pancreatic cancer.

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Lipidomics analysis of glycine-induced bacterial outer membrane vesicles SONG Jingyuan, QI Xiulei, GUO Huaizhong, HU Lianghai 2025, 43 (5):  547-555.  DOI: 10.3724/SP.J.1123.2024.10017 Abstract ( 87 )   HTML ( 12 )   PDF (4499KB) ( 28 )   Outer membrane vesicles (OMVs) are nanoparticles with double-phospholipid membrane structures that are secreted by gram-negative bacteria and carry a variety of bioactive substances from parental bacterial cells; consequently, OMVs serve as disease markers. Moreover, bacterial OMVs are potential anticancer- and antibacterial-drug carriers. While the addition of glycine during bacterial culturing promotes the secretion of bacterial OMVs, glycine-induced differences in the lipid compositions of such OMVs have not yet been reported. In this study, the key plasma membrane components of bacterial OMVs before and after glycine induction were analyzed using lipidomics. Bacterial OMVs were captured from bacterial-culture supernatants using an amphiphilic dendritic polymeric supramolecular probe. Two sets of enriched bacterial OMVs were characterized and their enrichment efficiencies determined, after which the numbers and purities of the OMVs within the samples were determined using a nanofluidic assay. Lipids were extracted using the methyl tert-butyl ether lipid-extraction method when consistent numbers were recorded. The lipid compositions of the bacterial OMVs before and after glycine induction were analyzed using an ultra-performance liquid chromatography-ion mobility spectrometry-quadrupole time-of-flight mass spectrometer (UPLC-IMS-QTOF-MS) and MS-DIAL software. Differential lipid species in the key plasma membranes of the bacterial OMVs following glycine induction were recorded along with their corresponding amounts. Detection was accomplished in positive-ion scanning mode using an ACQUITY UPLC BEH C18 column following UPLC-MS injection, and MSE mass-spectrometry data-acquisition mode. The lipid components in the two groups were determined by combining mass-spectrometric and software-analysis data, which revealed that the addition of glycine to the E. coli Nissle 1917 culture led to two-to-three-times higher concentrations of OMVs than observed for the untreated group under the same culturing and enrichment conditions. Particle numbers measured for the same volume revealed one-order-of-magnitude more bacterial OMVs after induction than before, with the treated group exhibiting slightly larger particles (on average); however, these particles were better dispersed and less likely to aggregate. The identified lipid components were categorized to determine the amount of each lipid type. Differentially expressed lipids were subsequently screened according to experimental conditions; significantly different expression levels were observed following glycine induction, with 820 lipids identified among the 10165 components detected. The lipid classes were ranked in order of quantity as: glycerolipids (GL), fatty acids (FA), sphingolipids (SP), glycerophospholipids (GP), saccharolipids (SL), and sterol lipids (ST), among which 463 GL lipid fractions (56.4% of all characterized lipids) were recorded. The ST lipid fraction contained the fewest members (10) and qualitative lipids were determined to make up 1.2% of the total. The addition of glycine to the E. coli culture was found to induce significant changes in the surface and internal lipid composition of the E. coli OMVs, with significantly more ceramide (Cer) and lysophosphatidyl choline (LPC), and significantly less bis(monoacylglycerol)phosphate (BMP) expressed. Partial triglyceride (TG) and sphingomyelin (SM) were irregularly expressed following glycine treatment, with equal amounts of up- and down-regulated lipids observed. This study provides a reference for subsequent in-depth studies into the lipid compositions of OMVs and their use as novel drug-delivery carriers. The expression of TGs and SM showed irregular changes, which is worthy of the next step of in-depth exploration of its regulatory mechanism, and the present study provides a certain reference for the subsequent in-depth study of the lipid composition of OMV and its use in the study of novel drug delivery carriers.