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 ◽  
...  

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.

2020 ◽  
Author(s):  
Rui Wei ◽  
Libo Zhao ◽  
Guanyi Kong ◽  
Xiang Liu ◽  
Shengtao Zhu ◽  
...  

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.


Small ◽  
2022 ◽  
pp. 2104470
Author(s):  
Sheng Yuan Leong ◽  
Hong Boon Ong ◽  
Hui Min Tay ◽  
Fang Kong ◽  
Megha Upadya ◽  
...  

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

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3156
Author(s):  
Daniel S. K. Liu ◽  
Flora M. Upton ◽  
Eleanor Rees ◽  
Christopher Limb ◽  
Long R. Jiao ◽  
...  

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.


2020 ◽  
Vol 58 (3) ◽  
pp. 408-415 ◽  
Author(s):  
Reza Soleimani ◽  
Julien Favresse ◽  
Tatiana Roy ◽  
Damien Gruson ◽  
Catherine Fillée

AbstractBackgroundThe correct identification of the macro-B12 interference (macroforms) is paramount to avoid potential erroneous clinical decisions. Our objectives were to determine whether immunoassays are affected by the presence of macro-B12 and to validate a polyethylene glycol (PEG) precipitation procedure to detect it.MethodsSixty-two serum samples obtained from healthy volunteers were analyzed to determine recovery and reference intervals (RIs) following PEG precipitation. Thereafter, 50 serum samples with very high levels of B12 (>1476 pmol/L) were randomly selected to search for macro-B12 interferences. Serum samples obtained from healthy volunteers and related PEG aliquots were analyzed on a Cobas® immunoassay. Patients’ samples were analyzed on both Cobas® and Architect® immunoassays. Finally, samples suspected to contain macro-B12 were analyzed by size-exclusion chromatography (SEC) to confirm the presence of macro-B12.ResultsRecovery and post-PEG RIs determined on a Cobas 8000® in healthy volunteers ranged from 68.3% to 108.4% and from 122.1 to 514.4 pmol/L, respectively. Fifteen samples (30%) were found to show macro-B12 while using the recovery criteria, and nine samples (18%) while using the post-PEG RI. The other immunoassay ran on the Architect i2000® was also affected by the presence of macro-B12. Size-exclusion chromatography studies confirmed the presence of macro-B12 (immunoglobulin-B12 complexes).ConclusionsThe prevalence of macro-B12 in elevated B12 samples is high. We suggest to systematically screen for the presence of macro-B12 with PEG precipitation procedure in samples with elevated B12 levels to avoid potential misdiagnosis or harmful clinical consequences.


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