{"doi":"10.1002/pmic.202100126","title":"Understanding extracellular vesicles","abstract":null,"journal":"PROTEOMICS","year":2021,"id":641170,"datarank":0.32958368660043297,"base_score":2.1972245773362196,"endowment":2.1972245773362196,"self_citation_contribution":0.32958368660043297,"citation_network_contribution":0.0,"self_endowment_contribution":0.32958368660043297,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":8,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":447800,"name":"Richard J. Simpson","orcid":"0000-0002-9834-0796","position":1,"is_corresponding":false},{"id":818124,"name":"David W. Greening","orcid":"0000-0001-7516-485X","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Understanding extracellular vesicles","abstract":"Extracellular vesicles (EVs) represent an important mode of intercellular communication, by selective packaging and transfer of biological information between cells. The advent of quantitative mass spectrometry-based proteomics, in conjunction with advances in molecular cell biology, and EV purification strategies, has contributed significantly to our improved characterization and understanding of the molecular composition and functionality of EVs. The field of EVs is a rapidly growing area of basic, applied, and biomedical research, with the identification of new types of EVs, their biology and functions, as well as the development of novel approaches to purify, and characterize them. This themed research topic has covered a number of cutting-edge discoveries, and importantly, how proteomics is advancing key questions in the field. Quantitative mass spectrometry-based proteomics holds the promise of identification, quantification, and validation of EV proteins, determining EV subtype-specific markers and biological insights in how EVs modulate and interact with target cells, directed towards biomedical research, therapeutic considerations, and clinical application. This topic has provided new data on EV subtypes, their biogenesis, influence on target cells, and considerations in their manufacturer and application in biomarker discovery. We predict an innovative and diverse future for expanding the application of mass spectrometry proteomics to discovery research, manufacturing, preclinical analyses, and clinical and therapeutic development in the EV community. We thank all contributors to this special issue research topic and the referees for their prompt and in-depth reviews. Composition and function of different EVs is influenced by their biogenesis. Taylor et al. (10.1002/pmic.202000091) investigated the biogenesis of plasma membrane EVs in the context of malignancy, revealing the subcellular localization and functional role of αII-spectrin in shed membrane EV vesiculation. Further, Carli et al. (10.1002/pmic.202000098) demonstrated the contribution of cancer stem cell marker DCLK1 influences small extracellular vesicle (sEV) biogenesis in a kinase-dependent manner in gastric cancer cells. Using a combination of functional in vitro assays, genetically modified cells, and quantitative proteome analysis, showed a specific role of DCLK1-kinase dependent cargo selection for sEVs, and its role as a regulator of signalling in gastric tumorigenesis. Importantly, this study revealed these kinase-dependent effects were EV subtype specific, providing important implications in the understanding of EV biology. Using flow cytometry and proteome analysis, the composition of a distinct subtype of EVs released by apoptotic cells (known as apoptotic bodies) was shown by Phan et al. (10.1002/pmic.13393) to be regulated by PANX1 channel. Indeed, using pharmacological inhibition and genetic disruption, perturbed the formation (DNA and nuclear protein composition) and size of these EVs. Generation and composition of EVs is influenced by media formulation. Garcia-Ceron et al. (10.1002/pmic.202000240) used an efficient and scalable size-based (non-centrifugation) EV isolation strategy and characterized the proteome of fungal-derived EVs from different growth media, demonstrating that media type influenced the proteome content, including known virulence regulators. This proteomic description suggests a role for fungal EVs in modulating host-pathogen interactions and expands our knowledge of plant pathogen EVs. Zhu et al. pmic.202000080 monitored the impact of culture conditions on stem cell-derived EV yield, potency, surface epitopes, and proteome composition (global EV proteome, gene ontology functional enrichment analyses, inclusion and exclusion markers of EVs), revealing important considerations in process optimization methods for the generation of EVs for development for therapeutic and clinical translation. Phillips et al. (10.1002/pmic.202000118) reviewed how the protein composition reflects EV heterogeneity, directly comparing the protein composition of different EV classes and subpopulations. The review also reflected on current limitations with size-based isolation approaches and highlights the development of affinity-based isolation tools as scalable methodologies to understand distinct EV subpopulations. Understanding interaction and uptake of EVs for cell-to-cell communication is important to define how EVs regulate phenotypic changes in target cells. Trigg et al. (10.1002/pmic.202000079) described optimization of a tractable cell culture model to study the mechanistic basis of sperm-EV interactions, specifically MFGE8 ligand and its cognate integrin receptor. Transfer of EV protein and microRNA cargo to mouse spermatozoa was assessed by biotinylation/affinity labelling, and confocal microscopy. Targeted mutation of MFGE8 provided direct insight into sperm-EV interaction through MFGE8 and RGD integrin domain. Here, this EV-binding interaction was shown to be important in underpinning mouse spermatozoa maturation. To understand how cellular reprogramming can influence the composition of EVs and their function, Shafiq et al. (10.1002/pmic.202000221) investigated how shed microvesicles (sMVs) composition is influenced following oncogenic H-Ras induced epithelial-mesenchymal transition (EMT). This study showed that sMVs following EMT promote cell migration, confer anchorage-independent growth, induce EMT in parental epithelial MDCK cells, and functionally contributed (via TGM2) in invasive capability of fibroblasts. Such findings highlight that cellular reprogramming following EMT influences sMV composition, and that this EV subtype play an important role in the EMT process. Reale et al. (10.1002/pmic.202000119) investigated how cell-derived and circulating (plasma-derived) small EVs (sEVs) from patients with distinct multiple myeloma stages regulate stromal cells to functionally modulate proliferation and migration following transfer. Various commercial approaches in sEV isolation and pre-analytical conditions (collection tube types, storage conditions) were assessed for sEV yield and marker enrichment, with proteome profiling revealing oncogenic cargo in circulating sEVs from malignant and pre-malignant patients. Specifically, this study highlights a protocol to generate morphologically-intact and functional sEVs capable of regulating stromal cells, and a model for the characterization of tumour-stromal cross-talk by sEV in multiple myeloma. Poh et al. (10.1002/pmic.202000210) investigated how sEVs (containing exosomes) from outer human embryo cells (stem cell-derived, trophectomderm) regulate endometrial function and our understanding of the molecular basis of implantation. Using highly-purified sEVs with biophysical and biochemical characterization, this study provides functional insights into their transfer to receptive endometrial cells, altered cell polarity, and proteins in sEVs implicated in embryo attachment, immune regulation, and antioxidant activity: processes critical for successful implantation. Given that EVs can be released by all cell types, and detected in most body fluids, understanding their composition in distinct tissue environments remains an active area of research. Claridge et al. (10.1002/pmic.202100026) investigated cardiac EVs (cEVs) as important mediators of intercellular signalling, using a unique isolation strategy employing direct treatment of heart using collagenase-based enzymatic perfusion and differential centrifugation. A striking finding, in direct comparison with other approaches mechanical-based approaches for isolation of EVs, in addition to heart tissue proteome, revealed comprehensive proteomic landscape of heart physiology, detection of EV markers, and depletion of blood and cardiac damage markers. cEVs contain marker proteins for various cardiac cell types, suggesting diverse cellular origin. This method of isolation and provides insight to understand EV composition and function in cardiac physiology and disease. Proteomic profiling of human uterine EVs by Rai et al. (10.1002/pmic.202000211) reveal dynamic regulation of key players of embryo implantation and fertility during menstrual cycle. This study investigated patient-derived total uterine lavage, soluble and EV fractions, and different quantitative mass spectrometry-based proteomic approaches (TMT/label free) to provide unique information into menstrual cycle phase (proliferative and secretory phases, associated with embryo implantation), in addition to fertility/infertility. Functionally, sEVs were shown to enhance antioxidant and invasion function in trophectoderm cells. This study provides novel insights into proteome reprogramming of sEVs and soluble secretome in uterine fluid, with potential to enhance embryo implantation and hence fertility. Proteomics is providing important insight into future directions of clinical application of EVs, including diagnosis (as non-invasive biomarkers via liquid biopsy) and therapeutic monitoring. Roberto et al. (10.1002/pmic.202000301) used patient-derived fibroblast EVs as a biomarker source with diagnostic and therapeutic potential for spinal muscular atrophy (SMA) revealing alterations in known markers implicated in the etiology of SMA. IGFBP3 was validated in multiple SMA patient derived cells, providing important insight into using EVs and proteomics in monitoring development of neurodegenerative diseases where defective mRNA metabolism may impact the proteome. Hermida-Nogueira et al. (10.1002/pmic.202000089) reviewed the implications of mass spectrometry and monitoring in transfusion medicine. This viewpoint highlights the potential of red blood cell- and platelet-derived EVs as a source of monitoring variables (i.e., storage lesions and possible transfusion adverse reactions) with clinical impact. Cho and colleagues (10.1002/pmic.202000094) review how proteomic profiling of oncogenic EVs is being applied in cancer detection and monitoring. The review covers important approaches in isolation of EVs for liquid biopsy, and utility of different clinical samples for EV isolation and clinical application. We believe this Special Issue provides a snapshot on the burgeoning field of EV classes and their subpopulations. With the continuing refinement of mass spectrometry-based proteomics [1], especially in the areas of quantitation [2-5], proteome coverage [6-10], single-cell or subtype analyses [11-13] and proteomic profiling with sequential window acquisition of all theoretical fragment ion spectra (SWATH) [4, 14]: we predict an exciting and diverse future for the EV field, especially in clinical and therapeutic applications. The authors declare no conflict of interest. David W. Greening, PhD, Head, Molecular Proteomics Baker Heart and Diabetes Institute, Australia Department of Biochemistry and Genetics La Trobe Institute for Molecular Science La Trobe University, Australia. Richard J. Simpson, PhD, FATSE Head, Distinguished Professor Department of Biochemistry and Genetics La Trobe Institute for Molecular Science La Trobe University, Australia.","is_dataset_classified":null,"base_score":2.1972245773362196,"endowment":2.1972245773362196,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"34138522","pmcid":null,"openalex_id":"https://openalex.org/W3171153171","authors":[],"funders":[],"total_grants":0,"fwci":0.2961,"citation_percentile":0.51724308,"influential_citations":0,"citation_trend":[{"year":2022,"count":2},{"year":2023,"count":1},{"year":2024,"count":1},{"year":2025,"count":2},{"year":2026,"count":2}],"oa_status":"bronze","license":"cc-by-nc","oa_locations":[{"url":"https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/pmic.202100126","host_type":"journal"},{"url":"https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/pmic.202100126","host_type":"publisher"},{"url":"https://onlinelibrary.wiley.com/doi/pdf/10.1002/pmic.202100126","host_type":"publisher"},{"url":"https://onlinelibrary.wiley.com/doi/full-xml/10.1002/pmic.202100126","host_type":"publisher"},{"url":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/pdf/10.1002/pmic.202100126","host_type":"publisher"},{"url":"https://doi.org/10.1002/pmic.202100126","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/34138522","host_type":"repository"},{"url":"https://figshare.com/articles/journal_contribution/Understanding_extracellular_vesicles/15095859","host_type":"repository"}],"fields_of_study":["Extracellular vesicles in disease","Cell Adhesion Molecules Research","Nanoplatforms for cancer theranostics"],"mesh_terms":["Extracellular Vesicles"],"keywords":["Proteomics","Extracellular vesicles","Computational biology","Identification (biology)","Biogenesis","Biomarker discovery","Quantitative proteomics","Function (biology)","Biology","Cell biology","Biochemistry"],"sdg_mappings":[{"sdg_number":0,"sdg_label":"Zero hunger"}],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-07T16:20:52.234766Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}