Deconstruction of Heterogeneity of Size-Dependent Exosome Subpopulations from Human Urine by Profiling N-Glycoproteomics and Phosphoproteomics Simultaneously

Zheng, Haoyang; Guan, Sheng; Wang, Xuantang; Zhao, Jiandong; Gao, Mingxia; Zhang, Xiangmin

doi:10.1021/acs.analchem.0c01572

Cited by 74 publications

(51 citation statements)

References 44 publications

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“…It is now widely accepted that EX populations differ depending on the cell type or biofluid of origin (12,13,56,57). To our knowledge, this is the first study to identify a significant difference in the mean and mode sizes of EX based on whether UC was performed before or after SEC.…”

Section: Discussionmentioning

confidence: 90%

A Comparison of Blood Plasma Exosome Enrichment Strategies for Proteomic Analysis

Turner¹,

Abeysinghe²,

Cheung³

et al. 2021

Preprint

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Proteomic analysis of exosomes (EX) poses a significant challenge. A ‘gold-standard’ method for plasma EX enrichment for downstream proteomic analysis is yet to be established. Our group has performed a comprehensive study of multi-dimensional enrichment methods to determine their efficiency for protein isolation. Methods were evaluated for their capacity to a) successfully isolate and enrich EX from blood plasma, b) minimise the presence of highly abundant plasma proteins, and c) result in the optimum representation of EX proteins by liquid chromatography tandem mass spectrometry (LC-MS/MS). Blood plasma from four animals (Bos taurus) of similar physical attributes and genetics were used. Three methods of EX enrichment were utilised: ultracentrifugation (UC), size-exclusion chromatography (SEC), and ultrafiltration (UF). These enrichment methods were combined to create four groups for methodological evaluation: UC+SEC, UC+SEC+UF, SEC+UC and SEC+UF. UC+SEC yielded the highest number of protein IDs. Plasma protein identification was the least in SEC+UC, but this method yielded the lowest number of protein IDs overall. UC+SEC+UF decreased EX protein ID and did not improve purity compared to UC+SEC. Our data suggest that the method and sequence of EX enrichment strategy impacts protein ID, which may influence the outcome of biomarker discovery studies.

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Section: Discussionmentioning

confidence: 90%

A Comparison of Blood Plasma Exosome Enrichment Strategies for Proteomic Analysis

Turner¹,

Abeysinghe²,

Cheung³

et al. 2021

Preprint

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show abstract

“…Further studies support this view. [150,151] However, the relationship between exosome size and their function/origin remains largely unclear and requires further investigation. In addition, microfluidic devices for separating sub-type exosomes are few in number and much effort needs to be made to provide technical support for this research.…”

Section: Separation Of Sub-population Exosomesmentioning

confidence: 99%

A Holistic Review of the State‐of‐the‐Art Microfluidics for Exosome Separation: An Overview of the Current Status, Existing Obstacles, and Future Outlook

Ding

Yang

Gao

et al. 2021

Small

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Exosomes, which form a class of extracellular vesicles (EVs), are membrane-bound lipid nanovesicles with sizes typically in the Exosomes, a class of small extracellular vesicles (30-150 nm), are secreted by almost all types of cells into virtually all body fluids. These small vesicles are attracting increasing research attention owing to their potential for disease diagnosis and therapy. However, their inherent heterogeneity and the complexity of bio-fluids pose significant challenges for their isolation. Even the "gold standard," differential centrifugation, suffers from poor yields and is time-consuming. In this context, recent developments in microfluidic technologies have provided an ideal system for exosome extraction and these devices exhibit some fascinating properties such as high speeds, good portability, and low sample volumes. In this review, the focus is on the state-ofthe-art microfluidic technologies for exosome isolation and highlight potential directions for future research and development by analyzing the challenges faced by the current strategies. range of 30-150 nm. [1] They are secreted by almost all cells into diverse bio-fluids, including blood, urine, breast milk, saliva, lymph, and cerebrospinal fluid. [2] The discovery of exosomes dates back to the 1980s, but for many years after their discovery, they were regarded as "dust". [3,4] However, recent studies have shown that they play a crucial role in intercellular communication. [5,6] The biogenesis of exosomes includes double invagination of membranes, formation of intracellular multivesicular bodies (MVBs), and the release of exosomes (Figure 1). The first invagination process (plasma-membrane budding) generates early-sorting endosomes (ESEs) that can develop into late-sorting endosomes (LSEs). The LSEs are invaginated once again to form MVBs containing intraluminal vesicles (ILVs). Finally, the MVBs fuse with the plasma membrane to release ILVs (exosomes). [7] After release, these exosomes are taken up by recipient cells via multiple processes, such as macropinocytosis, fusion with the cell membrane, clathrin-dependent endocytosis, and phagocytosis. [8] The exosomes uptaken can act either at the surface of the recipient cells or deliver functional cargo in their bulk, thus affecting recipient-cell behavior. [9] Thus, exosomes play a key role in various physiological and pathological processes, including mammalian reproduction and development, immune responses, and disease progression. [10] Exosomes are reported to exhibit many excellent characteristics for clinical applications (Figure 2). 1) They can reflect the real-time state of the original cell, as they are actively secreted by living cells. 2) They are abundant in virtually all biological fluids (up to 10 10 vesicles per mL). [11] 3) Exosomes have enriched contents, including cell-surface substances and cytoplasmic constituents (including proteins, nucleic acids, lipids, and metabolites). Furthermore, exosomes can not only protect enzyme-sensitive cargos from degradation, but also ...

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“…Another method is the size-exclusion chromatography (SEC) that separates EVs from different body fluids based on their hydrodynamic volume and molecular size [128]. The combination of two SEC columns (2D SEC) was recently shown to improve the isolation capacity of the different urinary exosome subpopulations [131]. Furthermore, the asymmetrical field-flow fractionation (AsFFF) allows the separation of EVs from plasma contaminants, such as lipoproteins (high-density and low-density lipoproteins), without subjecting the EVs to shear forces [132].…”

Section: New Technologies For Evs Isolationmentioning

confidence: 99%

Extracellular Vesicles as a Therapeutic Tool for Kidney Disease: Current Advances and Perspectives

Corrêa

Juncosa

Masereeuw

et al. 2021

IJMS

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Extracellular vesicles (EVs) have been described as important mediators of cell communication, regulating several physiological processes, including tissue recovery and regeneration. In the kidneys, EVs derived from stem cells have been shown to support tissue recovery in diverse disease models and have been considered an interesting alternative to cell therapy. For this purpose, however, several challenges remain to be overcome, such as the requirement of a high number of EVs for human therapy and the need for optimization of techniques for their isolation and characterization. Moreover, the kidney’s complexity and the pathological process to be treated require that EVs present a heterogeneous group of molecules to be delivered. In this review, we discuss the recent advances in the use of EVs as a therapeutic tool for kidney diseases. Moreover, we give an overview of the new technologies applied to improve EVs’ efficacy, such as novel methods of EV production and isolation by means of bioreactors and microfluidics, bioengineering the EV content and the use of alternative cell sources, including kidney organoids, to support their transfer to clinical applications.

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Deconstruction of Heterogeneity of Size-Dependent Exosome Subpopulations from Human Urine by Profiling N-Glycoproteomics and Phosphoproteomics Simultaneously

Cited by 74 publications

References 44 publications

A Comparison of Blood Plasma Exosome Enrichment Strategies for Proteomic Analysis

A Comparison of Blood Plasma Exosome Enrichment Strategies for Proteomic Analysis

A Holistic Review of the State‐of‐the‐Art Microfluidics for Exosome Separation: An Overview of the Current Status, Existing Obstacles, and Future Outlook

Extracellular Vesicles as a Therapeutic Tool for Kidney Disease: Current Advances and Perspectives

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