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.

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.

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 Analysis of Cerebrospinal Fluid Extracellular Vesicles by Proximity Extension Assay: A Comparative Study of Four Isolation Kits

2020 ◽  
Vol 21 (24) ◽  
pp. 9425
Author(s):  
Sebastian Sjoqvist ◽  
Kentaro Otake ◽  
Yoshihiko Hirozane
Keyword(s):  
Cerebrospinal Fluid ◽  
Extracellular Vesicles ◽  
Proteomic Analysis ◽  
Magnetic Beads ◽  
Biomarker Discovery ◽  
De Novo ◽  
Size Exclusion ◽  
Cell Regulation ◽  
Promising Source ◽  
Proximity Extension Assay

There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood–brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability.

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.

scholarly journals Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples

2015 ◽  
Vol 4 (1) ◽  
pp. 27369 ◽  
Author(s):  
Inés Lozano-Ramos ◽  
Ioana Bancu ◽  
Anna Oliveira-Tercero ◽  
María Pilar Armengol ◽  
Armando Menezes-Neto ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Urine Samples ◽  
Size Exclusion ◽  
Exclusion Chromatography
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