Analysis of Cerebrospinal Fluid Extracellular Vesicles by Proximity Extension Assay: A Comparative Study of Four Isolation Kits

There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood–brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability.

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A Comparative Proteomic Analysis of Extracellular Vesicles Associated With Lipotoxicity

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.735001 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yasuhiko Nakao ◽

Masanori Fukushima ◽

Amy S. Mauer ◽

Chieh-Yu Liao ◽

Anya Ferris ◽

...

Keyword(s):

Size Exclusion Chromatography ◽

Extracellular Vesicles ◽

Proteomic Analysis ◽

De Novo ◽

Size Exclusion ◽

Dependent Manner ◽

Exclusion Chromatography ◽

Acid Labile Subunit ◽

Molecular Patterns ◽

Gradient Separation

Extracellular vesicles (EVs) are emerging mediators of intercellular communication in nonalcoholic steatohepatitis (NASH). Palmitate, a lipotoxic saturated fatty acid, activates hepatocellular endoplasmic reticulum stress, which has been demonstrated to be important in NASH pathogenesis, including in the release of EVs. We have previously demonstrated that the release of palmitate-stimulated EVs is dependent on the de novo synthesis of ceramide, which is trafficked by the ceramide transport protein, STARD11. The trafficking of ceramide is a critical step in the release of lipotoxic EVs, as cells deficient in STARD11 do not release palmitate-stimulated EVs. Here, we examined the hypothesis that protein cargoes are trafficked to lipotoxic EVs in a ceramide-dependent manner. We performed quantitative proteomic analysis of palmitate-stimulated EVs in control and STARD11 knockout hepatocyte cell lines. Proteomics was performed on EVs isolated by size exclusion chromatography, ultracentrifugation, and density gradient separation, and EV proteins were measured by mass spectrometry. We also performed human EV proteomics from a control and a NASH plasma sample, for comparative analyses with hepatocyte-derived lipotoxic EVs. Size exclusion chromatography yielded most unique EV proteins. Ceramide-dependent lipotoxic EVs contain damage-associated molecular patterns and adhesion molecules. Haptoglobin, vascular non-inflammatory molecule-1, and insulin-like growth factor-binding protein complex acid labile subunit were commonly detected in NASH and hepatocyte-derived ceramide-dependent EVs. Lipotoxic EV proteomics provides novel candidate proteins to investigate in NASH pathogenesis and as diagnostic biomarkers for hepatocyte-derived EVs in NASH patients.

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Glycosylation of Cancer Extracellular Vesicles: Capture Strategies, Functional Roles and Potential Clinical Applications

Cells ◽

10.3390/cells10010109 ◽

2021 ◽

Vol 10 (1) ◽

pp. 109

Author(s):

Álvaro M. Martins ◽

Cátia C. Ramos ◽

Daniela Freitas ◽

Celso A. Reis

Keyword(s):

Extracellular Vesicles ◽

Biomarker Discovery ◽

De Novo ◽

Recipient Cell ◽

Cell Communication ◽

Cancer Biomarkers ◽

Functional Roles ◽

Molecular Information ◽

Glycosylation Pathway ◽

Organ Tropism

Glycans are major constituents of extracellular vesicles (EVs). Alterations in the glycosylation pathway are a common feature of cancer cells, which gives rise to de novo or increased synthesis of particular glycans. Therefore, glycans and glycoproteins have been widely used in the clinic as both stratification and prognosis cancer biomarkers. Interestingly, several of the known tumor-associated glycans have already been identified in cancer EVs, highlighting EV glycosylation as a potential source of circulating cancer biomarkers. These particles are crucial vehicles of cell–cell communication, being able to transfer molecular information and to modulate the recipient cell behavior. The presence of particular glycoconjugates has been described to be important for EV protein sorting, uptake and organ-tropism. Furthermore, specific EV glycans or glycoproteins have been described to be able to distinguish tumor EVs from benign EVs. In this review, the application of EV glycosylation in the development of novel EV detection and capture methodologies is discussed. In addition, we highlight the potential of EV glycosylation in the clinical setting for both cancer biomarker discovery and EV therapeutic delivery strategies.

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Faculty Opinions recommendation of Proteomic analysis of cerebrospinal fluid extracellular vesicles reveals synaptic injury, inflammation, and stress response markers in HIV patients with cognitive impairment.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.737043730.793570277 ◽

2020 ◽

Author(s):

Santosh Kumar

Keyword(s):

Cerebrospinal Fluid ◽

Cognitive Impairment ◽

Stress Response ◽

Extracellular Vesicles ◽

Proteomic Analysis ◽

Hiv Patients

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exRNA Signatures in Extracellular Vesicles and their Tumor-Lineage from Prostate Cancer

10.1101/2020.09.28.20190009 ◽

2020 ◽

Author(s):

Navneet Dogra ◽

Mehmet Eren Ahsen ◽

Edgar Gonzalez Kozlova ◽

Tzu-yi Chen ◽

kimaada allette ◽

...

Keyword(s):

Prostate Cancer ◽

Differential Expression ◽

Extracellular Vesicles ◽

Small Rna ◽

Tumor Tissue ◽

Biomarker Discovery ◽

De Novo ◽

Culture Media ◽

Tumor Resection ◽

Small Rna Sequencing

Circulating extracellular vesicles (EVs) present in the bodily fluids of patients with cancer may provide non-invasive access to the tumor tissue. Yet, the transcriptomic lineage of tumor-derived EVs before and after tumor-resection remains poorly understood. Here, we established 60 total small RNA-sequencing profiles from 17 aggressive prostate cancer (PCa) patients tumor and adjacent normal tissue, and EVs isolated from urine, serum, and cancer cell culture media. We interrogated the key satellite alteration in tumor-derived EVs and found that resection of tumor prostate tissue leads to differential expression of reactive oxygen species (ROS), P53 pathways, inflammatory/cytokines, oncogenes, and tumor suppressor genes in the EV nanosatellites. Furthermore, we provide a set of novel EV-specific RNA signature, which are present in cancer but are nonexistent in post-resection patients with undetectable cancer. Finally, using a de novo RNAseq assembly followed by characterization of the small RNA landscape, we found novel small RNA clusters (smRCs) in the EVs, which reside in the unannotated regions. Novel smRCs were orthogonally validated for their differential expression in the biomarker discovery cohort using RT-qPCR. We demonstrate that circulating tumor EVs provide a glimpse of the tumor tissue biology, resolving a major bottleneck in the current liquid biopsy efforts. Secretory vesicles appear to be playing a key role in non-canonical Wnt signaling and miRNA pathways, similar to the circulating tumor cells (CTCs), hence, we propose that such vesicles be called circulating tumor extracellular vesicles (CTEVs).

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Proteomic analysis of extracellular vesicles in cerebrospinal fluid of patients with Alzheimer’s disease

10.1101/2020.02.22.20026609 ◽

2020 ◽

Author(s):

Davide Chiasserini ◽

Irene Bijnsdorp ◽

Giovanni Bellomo ◽

Pier Luigi Orvietani ◽

Sander R. Piersma ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cerebrospinal Fluid ◽

Extracellular Vesicles ◽

Biomarker Discovery ◽

Neurodegenerative Disorder ◽

High Resolution Mass Spectrometry ◽

Biological Fluids ◽

Protein Profile ◽

Protein Markers

AbstractCerebrospinal fluid (CSF) contains different types of extracellular vesicles (EVs) with undisclosed biomarker potential for neurodegenerative diseases. The aims of the present study were: i) to compare the proteome EVs isolated using different ultracentrifugation speed ii) to preliminary explore the EVs proteome in a common neurodegenerative disorder, Alzheimer’s disease (AD) compared to neurological controls. CSF samples from control subjects and AD patients were pooled separately (15 mL) and subjected to ultracentrifugation (UC) at different speeds (20,000g and 100,000g) to isolate separate EV fractions (P20 and P100). The proteome was analysed using high-resolution mass spectrometry (LC-MS/MS) and comparisons were made using bioinformatic analysis. EVs isolated at 100,000g (P100) had a proteome consistent with vesicles secreted via an ESCRT-dependent mechanism, being highly enriched in alix (PDCD6IP), syntenin-1 (SDCBP) and TSG101. EVs isolated at 20,000g were substantially different, showing enrichment in cytoskeletal and cell adhesion molecules. The pools from patients diagnosed with AD showed a distinct protein profile of CSF EVs, with increased levels of ADAM10, SPON1, CH3IL1 and MDK in the P100 fraction. CSF EV offer a new potential biosource of protein markers for AD detection and a complementary framework to the analysis of whole biological fluids for biomarker discovery.

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Size-Exclusion Chromatography as a Technique for the Investigation of Novel Extracellular Vesicles in Cancer

Cancers ◽

10.3390/cancers12113156 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3156

Author(s):

Daniel S. K. Liu ◽

Flora M. Upton ◽

Eleanor Rees ◽

Christopher Limb ◽

Long R. Jiao ◽

...

Keyword(s):

Systematic Review ◽

Cancer Cells ◽

Size Exclusion Chromatography ◽

Extracellular Vesicles ◽

Biomarker Discovery ◽

Extracellular Vesicle ◽

Size Exclusion ◽

Isolation Method ◽

Isolation And Purification ◽

Exclusion Chromatography

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.

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Longitudinal Bottom-Up Proteomics of Serum, Serum Extracellular Vesicles, and Cerebrospinal Fluid Reveals Candidate Biomarkers for Early Detection of Glioblastoma in a Murine Model

Molecules ◽

10.3390/molecules26195992 ◽

2021 ◽

Vol 26 (19) ◽

pp. 5992

Author(s):

Francesco Greco ◽

Federica Anastasi ◽

Luca Fidia Pardini ◽

Marialaura Dilillo ◽

Eleonora Vannini ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Extracellular Vesicles ◽

Murine Model ◽

Biomarker Discovery ◽

Early Stage ◽

Protein Profile ◽

Survival Rates ◽

Non Invasive ◽

Tumor Stages ◽

Early Effects

Glioblastoma Multiforme (GBM) is a brain tumor with a poor prognosis and low survival rates. GBM is diagnosed at an advanced stage, so little information is available on the early stage of the disease and few improvements have been made for earlier diagnosis. Longitudinal murine models are a promising platform for biomarker discovery as they allow access to the early stages of the disease. Nevertheless, their use in proteomics has been limited owing to the low sample amount that can be collected at each longitudinal time point. Here we used optimized microproteomics workflows to investigate longitudinal changes in the protein profile of serum, serum small extracellular vesicles (sEVs), and cerebrospinal fluid (CSF) in a GBM murine model. Baseline, pre-symptomatic, and symptomatic tumor stages were determined using non-invasive motor tests. Forty-four proteins displayed significant differences in signal intensities during GBM progression. Dysregulated proteins are involved in cell motility, cell growth, and angiogenesis. Most of the dysregulated proteins already exhibited a difference from baseline at the pre-symptomatic stage of the disease, suggesting that early effects of GBM might be detectable before symptom onset.

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Combination of size-exclusion chromatography and ultracentrifugation improves the proteomic profiling of plasma derived small extracellular vesicles

10.21203/rs.3.rs-21362/v1 ◽

2020 ◽

Author(s):

Rui Wei ◽

Libo Zhao ◽

Guanyi Kong ◽

Xiang Liu ◽

Shengtao Zhu ◽

...

Keyword(s):

Size Exclusion Chromatography ◽

Extracellular Vesicles ◽

Proteomic Analysis ◽

Single Step ◽

Size Exclusion ◽

Systematic Evaluation ◽

Proteomic Profiling ◽

Exclusion Chromatography ◽

Associated Proteins ◽

No Gold Standard

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.

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L1CAM is not Associated with Extracellular Vesicles in Human Cerebrospinal Fluid or Plasma

10.1101/2020.08.12.247833 ◽

2020 ◽

Author(s):

Maia Norman ◽

Dmitry Ter-Ovanesyan ◽

Wendy Trieu ◽

Roey Lazarovits ◽

Emma J.K. Kowal ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Human Plasma ◽

Extracellular Vesicles ◽

Single Molecule ◽

Transmembrane Protein ◽

Gradient Centrifugation ◽

Opportunity To Learn ◽

Size Exclusion ◽

Potential Candidate ◽

Free Protein

AbstractNeuron-derived extracellular vesicles (NDEVs) present a tremendous opportunity to learn about the biochemistry of brain cells in living patients. L1CAM is a transmembrane protein expressed in neurons that is presumed to be found on NDEVs in human biofluids. Previous studies have used L1CAM immuno-isolation from human plasma to isolate NDEVs for neurodegenerative disease diagnostics. We developed a panel of ultrasensitive Single Molecule Array (Simoa) assays for known EV markers, as well as L1CAM, and applied it to study EVs in human plasma and cerebrospinal fluid (CSF). We fractionated plasma and CSF by size exclusion chromatography (SEC) and density gradient centrifugation (DGC) to separate EVs from free proteins. We observed that L1CAM did not elute in the EV fractions, but rather eluted in the free protein fractions. We found that L1CAM is present as a free protein in human plasma and CSF, possibly due to proteolytic cleavage and/or alternative splicing. We further demonstrate that the isoforms found in CSF and plasma are different. These data collectively establish that L1CAM in plasma is not EV associated and should therefore not be used for NDEV isolation. Importantly, the framework and tools described herein will allow for evaluation of other potential candidate markers for isolation of NDEVs.

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