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Towards optimised extracellular vesicle proteomics from cerebrospinal fluid.

ABSTRACT: The proteomic profile of extracellular vesicles (EVs) from cerebrospinal fluid (CSF) can reveal novel biomarkers for diseases of the brain. Here, we validate an ultrafiltration combined with size-exclusion chromatography (UF-SEC) method for isolation of EVs from canine CSF and probe the effect of starting volume on the EV proteomics profile. First, we performed a literature review of CSF EV articles to define the current state of art, discovering a need for basic characterisation of CSF EVs. Secondly, we isolated EVs from CSF by UF-SEC and characterised the SEC fractions by protein amount, particle count, transmission electron microscopy, and immunoblotting. Data are presented as mean ± standard deviation. Using proteomics, SEC fractions 3-5 were compared and enrichment of EV markers in fraction 3 was detected, whereas fractions 4-5 contained more apolipoproteins. Lastly, we compared starting volumes of pooled CSF (6 ml, 3 ml, 1 ml, and 0.5 ml) to evaluate the effect on the proteomic profile. Even with a 0.5 ml starting volume, 743 ± 77 or 345 ± 88 proteins were identified depending on whether 'matches between runs' was active in MaxQuant. The results confirm that UF-SEC effectively isolates CSF EVs and that EV proteomic analysis can be performed from 0.5 ml of canine CSF.

SUBMITTER: Kangas P

PROVIDER: S-EPMC10261101 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Towards optimised extracellular vesicle proteomics from cerebrospinal fluid.

Kangas Petra P Nyman Tuula A TA Metsähonkala Liisa L Burns Cameron C Tempest Robert R Williams Tim T Karttunen Jenni J Jokinen Tarja S TS

Scientific reports 20230612 1

The proteomic profile of extracellular vesicles (EVs) from cerebrospinal fluid (CSF) can reveal novel biomarkers for diseases of the brain. Here, we validate an ultrafiltration combined with size-exclusion chromatography (UF-SEC) method for isolation of EVs from canine CSF and probe the effect of starting volume on the EV proteomics profile. First, we performed a literature review of CSF EV articles to define the current state of art, discovering a need for basic characterisation of CSF EVs. Sec ...[more]

PMID: 37308520

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Project description:Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world-wide survey to ISEV members to assess methods considered 'best practices' for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.

Project description:The discovery of disease biomarkers, along with the use of "liquid biopsies" as a minimally invasive source of biomarkers, continues to be of great interest. In inflammatory diseases of the central nervous system (CNS), cerebrospinal fluid (CSF) is the most obvious biofluid source. Extracellular vesicles (EVs) are also present in CSF and are thought to be potential "biomarker treasure chests". However, isolating these CSF-derived EVs remains challenging. This small-scale pilot study developed and tested a protocol to enrich for CSF-EVs, both in relapsing remitting multiple sclerosis (RRMS) CSF and controls. These were subsequently compared, using an aptamer based proteomics array, SOMAscan™. EVs were enriched from RRMS patient (n = 4) and non-demyelinating control (idiopathic intracranial hypertension (IIH) (n = 3)) CSF using precipitation and mini size-exclusion chromatography (SEC). EV-enriched fractions were selected using pre-defined EV characteristics, including increased levels of tetraspanins. EVs and paired CSF were analysed by SOMAscan™, providing relative abundance data for 1128 proteins. CSF-EVs were characterised, revealing exosome-like features: rich in tetraspanins CD9 and CD81, size ~100 nm, and exosome-like morphology by TEM. Sufficient quantities of, SOMAscan™ compatible, EV material was obtained from 5 ml CSF for proteomics analysis. Overall, 348 and 580 proteins were identified in CSF-EVs and CSF, respectively, of which 50 were found to be significantly (t-test) and exclusively enriched in RRMS CSF-EVs. Selected proteins, Plasma kallikrein and Apolipoprotein-E4, were further validated by western blot and appeared increased in CSF-EVs compared to CSF. Functional enrichment analysis of the 50 enriched proteins revealed strong associations with biological processes relating to MS pathology and also extracellular regions, consistent with EV enrichment. This pilot study demonstrates practicality for EV enrichment in CSF derived from patients with MS and controls, allowing detailed analysis of protein profiles that may offer opportunities to identify novel biomarkers and therapeutic approaches in CNS inflammatory diseases.

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Towards optimised extracellular vesicle proteomics from cerebrospinal fluid.

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Towards optimised extracellular vesicle proteomics from cerebrospinal fluid.

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