Biomaterial Database

Ultrafiltration combing with phospholipid affinity-based isolation for metabolomic profiling of urinary extracellular vesicles. 2021

Doudou Lou, and Yong Wang, and Qinsi Yang, and Liang Hu, and Qingfu Zhu

Eye Hospital, School of Ophthalmology & Optometry, School of Biomedical Engineering, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China.

Recent years have seen the field of extracellular vesicle (EV) studies burgeoning. This is mainly because EV constituents including nucleic acid, proteins, lipids, and metabolites are promising sources towards disease biomarker discovery. However, EV study remains challenging due to the complexity of biofluids as well as technical limitations during sample preparation. Here, we proposed a simple method combing ultrafiltration (UF) and phospholipid affinity to collect high purity EVs from 30 mL of urine sample for their metabolomic profiling. Ultracentrifugation (UC) for EV isolation was applied as a reference method. Western blot (WB) analysis, nanoparticles tracking analysis (NTA) and electron microscopy (EM) were used to assess EV protein markers and to characterize vesicle size and morphology. The results revealed that more than 1010 EV particles could be isolated from a 30 mL urine sample by the proposed method, and the resulting EVs carry specific protein markers and had a typical "cup shape" morphology. This suggests that our method is suitable for EV isolation and can provide sufficient EV quantity to ensure downstream analysis. Further untargeted metabolomic profiling of isolated EVs by UHPLC-QTOF-MS detected 433 metabolites by our methods and 432 metabolites by UC with a MS/MS similarity score greater than 0.7. We then applied the lipid metabolites-targeted method using UHPLC-QTrap-MS with the MRM mode, which successfully detected 467 compounds from urine EVs. This indicates that UF integrating phospholipid affinity is a reliable method for metabolic analysis of urinary EVs, which holds the potential for EV clinical application towards biomarker investigation from their metabolites.

UI	MeSH Term	Description	Entries
D010743	Phospholipids	Lipids containing one or more phosphate groups, particularly those derived from either glycerol (phosphoglycerides see GLYCEROPHOSPHOLIPIDS) or sphingosine (SPHINGOLIPIDS). They are polar lipids that are of great importance for the structure and function of cell membranes and are the most abundant of membrane lipids, although not stored in large amounts in the system.	Phosphatides,Phospholipid
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000067128	Extracellular Vesicles	Membrane limited structures derived from cell membranes and cytoplasmic material, and released into EXTRACELLULAR SPACE. They circulate through the EXTRACELLULAR FLUID and through the peripheral blood in the MICROVASCULATURE where cells, much larger, cannot, thereby affecting a variety of intercellular communication processes.	Apoptotic Bodies,Exovesicles,Apoptotic Body,Bodies, Apoptotic,Body, Apoptotic,Exovesicle,Extracellular Vesicle,Vesicle, Extracellular,Vesicles, Extracellular
D014025	Titanium	A dark-gray, metallic element of widespread distribution but occurring in small amounts with atomic number, 22, atomic weight, 47.867 and symbol, Ti; specific gravity, 4.5; used for fixation of fractures.
D014462	Ultrafiltration	The separation of particles from a suspension by passage through a filter with very fine pores. In ultrafiltration the separation is accomplished by convective transport; in DIALYSIS separation relies instead upon differential diffusion. Ultrafiltration occurs naturally and is a laboratory procedure. Artificial ultrafiltration of the blood is referred to as HEMOFILTRATION or HEMODIAFILTRATION (if combined with HEMODIALYSIS).
D014556	Urine	Liquid by-product of excretion produced in the kidneys, temporarily stored in the bladder until discharge through the URETHRA.
D053719	Tandem Mass Spectrometry	A mass spectrometry technique using two (MS/MS) or more mass analyzers. With two in tandem, the precursor ions are mass-selected by a first mass analyzer, and focused into a collision region where they are then fragmented into product ions which are then characterized by a second mass analyzer. A variety of techniques are used to separate the compounds, ionize them, and introduce them to the first mass analyzer. For example, for in GC-MS/MS, GAS CHROMATOGRAPHY-MASS SPECTROMETRY is involved in separating relatively small compounds by GAS CHROMATOGRAPHY prior to injecting them into an ionization chamber for the mass selection.	Mass Spectrometry-Mass Spectrometry,Mass Spectrometry Mass Spectrometry,Mass Spectrometry, Tandem
D055432	Metabolomics	The systematic identification and quantitation of all the metabolic products of a cell, tissue, organ, or organism under varying conditions. The METABOLOME of a cell or organism is a dynamic collection of metabolites which represent its net response to current conditions.	Metabonomics,Metabolomic,Metabonomic
D055442	Metabolome	The dynamic collection of metabolites which represent a cell's or organism's net metabolic response to current conditions.	Metabolic Profile,Metabolic Profiles,Metabolomes,Profile, Metabolic,Profiles, Metabolic