Dataset Information

Proteomics, phosphoproteomics and miRNA analysis of circulating extracellular vesicles through automated and high-throughput isolation

ABSTRACT: Here, we introduce a magnetic bead-based EV enrichment approach (EVrich) for automated and high-throughput processing of urine samples. Parallel enrichments can be performed in 96-well plates for downstream cargo analysis, including EV characterization, miRNA, proteomics and phosphoproteomics analysis. We applied the instrument to a cohort of clinical urine samples to achieve reproducible identification of over 47,562 unique EV peptides and 4,327 EV proteins in each one-ml urine sample. Quantitative phosphoproteomicsrevealed 186 unique phosphopeptides corresponding to 77 proteins that were significantly elevated in prostate cancer patients.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine

DISEASE(S): Prostate Adenocarcinoma

SUBMITTER: Hao Zhang

LAB HEAD: W. Andy Tao

PROVIDER: PXD033595 | Pride | 2022-08-12

REPOSITORIES: Pride

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Publications

Proteomics, Phosphoproteomics and Mirna Analysis of Circulating Extracellular Vesicles through Automated and High-Throughput Isolation.

Zhang Hao H Cai Yu-Han YH Ding Yajie Y Zhang Guiyuan G Liu Yufeng Y Sun Jie J Yang Yuchen Y Zhan Zhen Z Iliuk Anton A Gu Zhongze Z Gu Yanhong Y Tao W Andy WA

Cells 20220629 13

Extracellular vesicles (EVs) play an important role in the diagnosis and treatment of diseases because of their rich molecular contents involved in intercellular communication, regulation, and other functions. With increasing efforts to move the field of EVs to clinical applications, the lack of a practical EV isolation method from circulating biofluids with high throughput and good reproducibility has become one of the biggest barriers. Here, we introduce a magnetic bead-based EV enrichment app ...[more]

PMID: 35805153

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Project description:Objectives: Secretion of extracellular vesicles (EV) and associated micro-RNAs (miR) is altered during cellular stress and may serve as biomarkers of organ injury. We hypothesized that measuring changes in urinary levels of EV and miR will predict the onset of acute kidney injury in cardiac surgery patients. Design: Predictive accuracy biomarker study performed in the cohort of the REVAKI-2 trial Setting: Single center ICU between September 2015 and September 2018 Interventions: Intravenous sildenafil citrate 12.5 mg kg-1 over 150 min or dextrose 5% at the commencement of surgery. Measurements and main results: Urine samples were collected before and 24 hours after the procedure from 93 cardiac surgery patients. Urine EV concentrations and size distribution were assessed using NanoSight. EV derivation and levels were measured using flow cytometry. Samples from 10 selected patients were sequenced to detect differentially expressed miR. Verification was performed with advanced TaqMan assays in samples from all patients. Urine EV concentrations significantly increased in patients with AKI after surgery, with the percentage of EV positive for aquaporin-2 and β1-integrin also increasing. Pre surgery podocalyxin-positive EV were significantly lower, and β1-integrin EV werehigher in patients with AKI. The levels of the former correlated with the severity of theinjury. miR-125a-5p was expressed at higher levels in urine from patients with AKI stage 2/3. Levels of miR-10a-5p decreased after surgery in AKI patients; its levels correlated with the severity of the injury. Preoperative levels of podocalyxin EVs and miR-125a-5p had moderate AKI predictive value and, in a logistic model together with ICU lactate levels, offered good (AUC = 80.9%) AKI prediction. Conclusions: Lower levels of podocalyxin-positive EV at baseline predict the severity of post-surgery AKI. Urine EV concentrations and miR expression offer excellent predictive accuracy when combined with commonly measured biomarkers.

Project description:Tumor cells dissociate from the primary site and enter into systemic circulation (circulating tu-mor cells, CTCs) either alone or Tumor cells dissociate from the primary site and enter into systemic circulation (circulating tu-mor cells, CTCs) either alone or as tumor microemboli (clusters); the latter having an increased predisposition towards forming distal metastases than single CTCs. The formation of clusters is, in part, created by contacts between cell–cell junction proteins and/or cytokine receptor pairs with other cells such as neutrophils, platelets, fibroblasts, etc. In the present study, we provide evidence for an extravesicular (EV) mode of communication between pancreatic cancer CTCs and neutrophils. Our results suggest that the EV proteome of CTCs contain signaling proteins that can modulate degranulation and granule mobilization in neutrophils and, also, contain tis-sue plasminogen activator and other proteins that can regulate cluster formation. By exposing naïve neutrophils to EVs isolated from CTCs, we further show how these changes are modulated in a dynamic fashion indicating evidence for a deeper EV based remodulatory effect on com-panion cells in clusters.as tumor microemboli (clusters); the latter having an increased predisposition towards forming distal metastases than single CTCs. The formation of clusters is, in part, created by contacts between cell–cell junction proteins and/or cytokine receptor pairs with other cells such as neutrophils, platelets, fibroblasts, etc. In the present study, we provide evidence for an extravesicular (EV) mode of communication between pancreatic cancer CTCs and neutrophils. Our results suggest that the EV proteome of CTCs contain signaling proteins that can modulate degranulation and granule mobilization in neutrophils and, also, contain tis-sue plasminogen activator and other proteins that can regulate cluster formation. By exposing naïve neutrophils to EVs isolated from CTCs, we further show how these changes are modulated in a dynamic fashion indicating evidence for a deeper EV based remodulatory effect on com-panion cells in clusters.

Dataset Information

Proteomics, phosphoproteomics and miRNA analysis of circulating extracellular vesicles through automated and high-throughput isolation

Publications

Proteomics, Phosphoproteomics and Mirna Analysis of Circulating Extracellular Vesicles through Automated and High-Throughput Isolation.

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