Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences after Differential Ultracentrifugation and ExoQuick™ Isolation

Abstract

Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuick™ in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuick™. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuick™ compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs’ protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached.

Isolation and characterization of extracellular vesicles in saliva of children with asthma

“…This method results in a greater yield of EVs in comparison with other methods, but with lower purity. EQ samples contain EVs of varying sizes and high portions of salts, polymers, and other contaminants such as lipoproteins [ 48 ] , although our TEM analysis demonstrated there was very little lipoprotein contamination in the EVs isolated from CFS. There was, however, carryover of the EQ reagent Supplementary Figure 2 .…”

Proteomic profiling in extracellular vesicles for cancer detection and monitoring

“…Some commercial kits provide polymer‐based precipitation for rapid and simple exosome isolation in a small volume of body fluid samples [144]. However, tumor‐derived EVs isolated by DUC and polymer‐based precipitation methods have different protein expression profiles based on 2D‐DIGE and MALDI‐TOF‐MS [156]. Notably, compared with DUC that is the gold standard, the application of polymer‐based precipitation in EV isolation is still controversial.…”

“…tumor-derived EVs isolated by DUC and polymer-based precipitation methods have different protein expression profiles based on 2D-DIGE and MALDI-TOF-MS[156]. Notably, compared with DUC that is the gold standard, the application of polymer-based precipitation in EV isolation is still controversial.…”

Correction: Gemoll et al. Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences after Differential Ultracentrifugation and ExoQuick™ Isolation. J. Clin. Med. 2020, 9, 1429

“…In the original version of our article [ 1 ], insufficient acknowledgement was given for the proteomic experiments, sample selection, analysis of results and drafting of the manuscript. We apologize for the original error and any inconvenience caused by the omission of Sarah Strohkamp as an author.…”

“…We have found two inadvertent errors in our paper published in the Journal of Clinical Medicine [ 1 ].…”