scholarly journals Combination of size-exclusion chromatography and ultracentrifugation improves the proteomic profiling of plasma derived small extracellular vesicles

2020 ◽  
Author(s):  
Rui Wei ◽  
Libo Zhao ◽  
Guanyi Kong ◽  
Xiang Liu ◽  
Shengtao Zhu ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Proteomic Analysis ◽  
Single Step ◽  
Size Exclusion ◽  
Systematic Evaluation ◽  
Proteomic Profiling ◽  
Exclusion Chromatography ◽  
Associated Proteins ◽  
No Gold Standard

Abstract Background: Circulating small extracellular vesicles (sEVs) and its associated proteins are of great interest in the early detection of many diseases. However, there is no gold standard for plasma sEVs isolation, especially for proteomic profiling which could be largely affected by contamination such as lipoproteins. Previous studies suggested combinations of different sEVs isolation methods could improve the purity of the isolated fractions. Nevertheless, there is no systematic evaluation of size-exclusion chromatography (SEC), ultracentrifugation (UC) and their combination in a proteomic perspective. Results: Here we exhibited that SEC+UC showed comparable recovery of sEVs with higher purity in contrast to single-step UC or SEC isolation. In our proteomic analysis, there are 992 protein species identified in the sEVs fractions isolated by SEC+UC, much more than the sEVs fractions isolated by UC (453) or SEC (682) alone. As compared to Vesiclepedia and Exocarta databases, SEC+UC kept 584 previously identified sEV-associated proteins and 360 other proteins. Furthermore, detailed analysis suggested that sEV-associated proteins, such as CD9, CD81 and ITGB1, showed the better protein rank in SEC+UC group than UC group and SEC group. Lipoproteins, the most common contamination in sEVs proteomic analysis, along with other free-floating proteins in the plasma, were largely removed in SEC+UC. Conclusions: We suggested that combining SEC with UC could significantly improve the performance of mass-spectrum (MS)-based proteomic profiling in analyzing plasma-derived sEVs.

scholarly journals Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jik-Han Jung ◽  
Woojin Back ◽  
Junyong Yoon ◽  
Hyeonjeong Han ◽  
Ka-Won Kang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Plasma ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Soluble Proteins ◽  
Single Step ◽  
Size Exclusion ◽  
Design Parameters ◽  
Exclusion Chromatography ◽  
Porous Beads

AbstractIsolation of pure extracellular vesicles (EVs), especially from blood, has been a major challenge in the field of EV research. The presence of lipoproteins and soluble proteins often hinders the isolation of high purity EVs upon utilization of conventional separation methods. To circumvent such problems, we designed a single-step dual size-exclusion chromatography (dSEC) column for effective isolation of highly pure EVs from bone marrow derived human plasma. With an aim to select appropriate column design parameters, we analyzed the physiochemical properties of the major substances in bone marrow derived plasma, which include EVs, lipoproteins, and soluble proteins. Based on these findings, we devised a novel dSEC column with two different types of porous beads sequentially stacked each other for efficient separation of EVs from other contaminants. The newly developed dSEC columns exhibited better performance in isolating highly pure EVs from AML plasma in comparison to conventional isolation methods.

scholarly journals Single-step isolation of extracellular vesicles by size-exclusion chromatography

2014 ◽  
Vol 3 (1) ◽  
pp. 23430 ◽  
Author(s):  
Anita N. Böing ◽  
Edwin van der Pol ◽  
Anita E. Grootemaat ◽  
Frank A. W. Coumans ◽  
Auguste Sturk ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Single Step ◽  
Size Exclusion ◽  
Exclusion Chromatography

scholarly journals Human Serum Extracellular Vesicle Proteomic Profile Depends on the Enrichment Method Employed

2021 ◽  
Vol 22 (20) ◽  
pp. 11144
Author(s):  
Mikel Azkargorta ◽  
Ibon Iloro ◽  
Iraide Escobes ◽  
Diana Cabrera ◽  
Juan M. Falcon-Perez ◽  
...  
Keyword(s):  
Human Serum ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Membrane Attack Complex ◽  
Protein Detection ◽  
Size Exclusion ◽  
Blood Proteins ◽  
Proteomic Profiling ◽  
Serum Samples ◽  
Exclusion Chromatography

The proteomic profiling of serum samples supposes a challenge due to the large abundance of a few blood proteins in comparison with other circulating proteins coming from different tissues and cells. Although the sensitivity of protein detection has increased enormously in the last years, specific strategies are still required to enrich less abundant proteins and get rid of abundant proteins such as albumin, lipoproteins, and immunoglobulins. One of the alternatives that has become more promising is to characterize circulating extracellular vesicles from serum samples that have great interest in biomedicine. In the present work, we enriched the extracellular vesicles fraction from human serum by applying different techniques, including ultracentrifugation, size-exclusion chromatography, and two commercial precipitation methods based on different mechanisms of action. To improve the performance and efficacy of the techniques to promote purity of the preparations, we have employed a small volume of serum samples (<100 mL). The comparative proteomic profiling of the enriched preparations shows that ultracentrifugation procedure yielded a larger and completely different set of proteins than other techniques, including mitochondrial and ribosome related proteins. The results showed that size exclusion chromatography carries over lipoprotein associated proteins, while a polymer-based precipitation kit has more affinity for proteins associated with granules of platelets. The precipitation kit that targets glycosylation molecules enriches differentially protein harboring glycosylation sites, including immunoglobulins and proteins of the membrane attack complex.

scholarly journals A Comparative Proteomic Analysis of Extracellular Vesicles Associated With Lipotoxicity

2021 ◽  
Vol 9 ◽  
Author(s):  
Yasuhiko Nakao ◽  
Masanori Fukushima ◽  
Amy S. Mauer ◽  
Chieh-Yu Liao ◽  
Anya Ferris ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Proteomic Analysis ◽  
De Novo ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Exclusion Chromatography ◽  
Acid Labile Subunit ◽  
Molecular Patterns ◽  
Gradient Separation

Extracellular vesicles (EVs) are emerging mediators of intercellular communication in nonalcoholic steatohepatitis (NASH). Palmitate, a lipotoxic saturated fatty acid, activates hepatocellular endoplasmic reticulum stress, which has been demonstrated to be important in NASH pathogenesis, including in the release of EVs. We have previously demonstrated that the release of palmitate-stimulated EVs is dependent on the de novo synthesis of ceramide, which is trafficked by the ceramide transport protein, STARD11. The trafficking of ceramide is a critical step in the release of lipotoxic EVs, as cells deficient in STARD11 do not release palmitate-stimulated EVs. Here, we examined the hypothesis that protein cargoes are trafficked to lipotoxic EVs in a ceramide-dependent manner. We performed quantitative proteomic analysis of palmitate-stimulated EVs in control and STARD11 knockout hepatocyte cell lines. Proteomics was performed on EVs isolated by size exclusion chromatography, ultracentrifugation, and density gradient separation, and EV proteins were measured by mass spectrometry. We also performed human EV proteomics from a control and a NASH plasma sample, for comparative analyses with hepatocyte-derived lipotoxic EVs. Size exclusion chromatography yielded most unique EV proteins. Ceramide-dependent lipotoxic EVs contain damage-associated molecular patterns and adhesion molecules. Haptoglobin, vascular non-inflammatory molecule-1, and insulin-like growth factor-binding protein complex acid labile subunit were commonly detected in NASH and hepatocyte-derived ceramide-dependent EVs. Lipotoxic EV proteomics provides novel candidate proteins to investigate in NASH pathogenesis and as diagnostic biomarkers for hepatocyte-derived EVs in NASH patients.

Microfluidic Size Exclusion Chromatography (μSEC) for Extracellular Vesicles and Plasma Protein Separation

Small ◽  
2022 ◽  
pp. 2104470
Author(s):  
Sheng Yuan Leong ◽  
Hong Boon Ong ◽  
Hui Min Tay ◽  
Fang Kong ◽  
Megha Upadya ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Plasma Protein ◽  
Protein Separation ◽  
Size Exclusion ◽  
Exclusion Chromatography

scholarly journals Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles’ characteristics compared to precipitating agents

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Ana Gámez-Valero ◽  
Marta Monguió-Tortajada ◽  
Laura Carreras-Planella ◽  
Marcel·la Franquesa ◽  
Katrin Beyer ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Exclusion Chromatography

scholarly journals Size-Exclusion Chromatography as a Technique for the Investigation of Novel Extracellular Vesicles in Cancer

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3156
Author(s):  
Daniel S. K. Liu ◽  
Flora M. Upton ◽  
Eleanor Rees ◽  
Christopher Limb ◽  
Long R. Jiao ◽  
...  
Keyword(s):  
Systematic Review ◽  
Cancer Cells ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Biomarker Discovery ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Isolation Method ◽  
Isolation And Purification ◽  
Exclusion Chromatography

Cancer cells release extracellular vesicles, which are a rich target for biomarker discovery and provide a promising mechanism for liquid biopsy. Size-exclusion chromatography (SEC) is an increasingly popular technique, which has been rediscovered for the purposes of extracellular vesicle (EV) isolation and purification from diverse biofluids. A systematic review was undertaken to identify all papers that described size exclusion as their primary EV isolation method in cancer research. In all, 37 papers were identified and discussed, which showcases the breadth of applications in which EVs can be utilised, from proteomics, to RNA, and through to functionality. A range of different methods are highlighted, with Sepharose-based techniques predominating. EVs isolated using SEC are able to identify cancer cells, highlight active pathways in tumourigenesis, clinically distinguish cohorts, and remain functionally active for further experiments.

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