Assessment of urine sample collection and processing variables for extracellular vesicle-based proteomics.

Extracellular vesicles (EVs) in urine are a promising source for developing non-invasive biomarkers. However, urine concentration and content are highly variable and dynamic, and actual urine collection and handling often is nonideal. Furthermore, patients such as those with prostate diseases have challenges in sample collection due to difficulties in holding urine at designated time points. Here, we simulated the actual situation of clinical sample collection to examine the stability of EVs in urine under different circumstances, including urine collection time and temporary storage temperature, as well as daily urine sampling under different diet conditions. EVs were isolated using functionalized EVtrap magnetic beads and characterized by nanoparticle tracking analysis (NTA), western blotting, electron microscopy, and mass spectrometry (MS). EVs in urine remained relatively stable during temporary storage for 6 hours at room temperature and for 12 hours at 4 °C, while significant fluctuations were observed in EV amounts from urine samples collected at different time points from the same individuals, especially under certain diets. Sample normalization with creatinine reduced the coefficient of variation (CV) values among EV samples from 17% to approximately 6% and facilitated downstream MS analyses. Finally, based on the results, we applied them to evaluate potential biomarker panels in prostate cancer by data-independent acquisition (DIA) MS, presenting the recommendation that can facilitate biomarker discovery with nonideal handling conditions.

A fast, easy, cost-free method to remove excess dye or drug from small extracellular vesicle solution.

Tracking small extracellular vesicles (sEVs), such as exosomes, requires staining them with dyes that penetrate their lipid bilayer, a process that leaves excess dye that needs to be mopped up to achieve high specificity. Current methods to remove superfluous dye have limitations, among them that they are time-intensive, carry the risk of losing sample and can require specialized equipment and materials. Here we present a fast, easy-to-use, and cost-free protocol for cleaning excess dye from stained sEV samples by adding their parental cells to the mixture to absorb the extra dye much like sponges do. Since sEVs are considered a next-generation drug delivery system, we further show the success of our approach at removing excess chemotherapeutic drug, daunorubicin, from the sEV solution.

Recombinant extracellular vesicles as biological reference material for method development, data normalization and assessment of (pre-)analytical variables.

The diagnostic and therapeutic use of extracellular vesicles (EV) is under intense investigation and may lead to societal benefits. Reference materials are an invaluable resource for developing, improving and assessing the performance of regulated EV applications and for quantitative and objective data interpretation. We have engineered recombinant EV (rEV) as a biological reference material. rEV have similar biochemical and biophysical characteristics to sample EV and function as an internal quantitative and qualitative control throughout analysis. Spiking rEV in bodily fluids prior to EV analysis maps technical variability of EV applications and promotes intra- and inter-laboratory studies. This protocol, which is an Extension to our previously published protocol (Tulkens et al., 2020), describes the production, separation and quality assurance of rEV, their dilution and addition to bodily fluids, and the detection steps based on complementary fluorescence, nucleic acid and protein measurements. We demonstrate the use of rEV for method development, data normalization and assessment of pre-analytical variables. The protocol can be adopted by researchers with standard laboratory and basic EV separation/characterization experience and requires ~4-5 d.

Assessment of separation methods for extracellular vesicles from human and mouse brain tissues and human cerebrospinal fluids.

Extracellular vesicle (EV) is a unified terminology of membrane-enclosed vesicular species ubiquitously secreted by almost every cell type and present in all body fluids. They carry a cargo of lipids, metabolites, nucleic acids and proteins for their clearance from cells as well as for cell-to-cell communications. The exact composition of EVs and their specific functions are not well understood due to the underdevelopment of the separation protocols, especially those from the central nervous system including animal and human brain tissues as well as cerebrospinal fluids, and the low yield of proteins in the separated EVs. To understand their exact molecular composition and their functional roles, development of the reliable protocols for EV separation is necessary. Here we report the methods for EV separation from human and mouse unfixed frozen brain tissues by a sucrose step gradient ultracentrifugation method, and from human cerebrospinal fluids by an affinity capture method. The separated EVs were assessed for morphological, biophysical and proteomic properties of separated EVs by nanoparticle tracking analysis, transmission electron microscopy, and labeled and label-free mass spectrometry for protein profiling with step-by-step protocols for each assessment.

Association of Extracellular Vesicle Protein Cargo with Race and Clinical Markers of Mortality.

Differential mortality rates remain a significant health disparity in the United States, suggesting the need to investigate novel potential molecular markers associated with mortality. Extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic bodies, are lipid-bound vesicles secreted by cells into the circulation. EVs mediate intercellular communication by shuttling functional signaling molecules as cargo. EV characteristics by race in the context of mortality risk factors have not been described. We isolated plasma EVs from a cross-sectional cohort of African Americans (AA) and whites and found no significant differences in EV size, distribution or concentration between race or by sex. However, EV cargo showed increased levels of phospho-p53, total p53, cleaved caspase 3, ERK1/2 and phospho-AKT in white individuals compared to AAs. phospho-IGF-1R levels were significantly higher in females compared to males. EV concentration was significantly associated with several clinical mortality risk factors: high-sensitivity C-reactive protein (hsCRP), homeostatic model assessment of insulin resistance (HOMA-IR), alkaline phosphatase, body mass index, waist circumference and pulse pressure. The association of EV proteins with mortality markers were dependent on race. These data suggest that EV cargo can differ by race and sex and is associated with mortality risk factors.

Assessment of Extracellular Vesicles Purity Using Proteomic Standards.

The increasing interest in extracellular vesicles (EVs) research is fueled by reports indicating their unique role in intercellular communication and potential connection to the development of common human diseases. The unique role assumes unique protein and nucleic acid cargo. Unfortunately, accurate analysis of EVs cargo faces a challenge of EVs isolation. Generally used isolation techniques do not separate different subtypes of EVs and even more, poorly separate EVs from non-EVs contaminants. Further development of EVs isolation protocols urgently needs a quantitative method of EVs purity assessment. We report here that multiple reaction monitoring assay using internal standards carrying peptides for quantification of EVs and non-EVs proteins is a suitable approach to assess purity of EVs preparations. As a first step in potential standardization of EVs isolation, we have evaluated polymer-based precipitation techniques and compared them to traditional ultracentrifugation protocol.