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

Abstract

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.

A shared, stochastic pathway mediates exosome protein budding along plasma and endosome membranes

“…We began our cargo-based analysis of exosome biogenesis by identifying the most highly-enriched exosome cargo proteins in a model human cell line, HEK293. Of the many cargo proteins that have been reported, CD9, CD63, and CD81 were the first proteins shown to be enriched in exosomes (46,100), and as a result have been used as exosome marker proteins for more than 30 years (101)(102)(103). To determine whether these three proteins are more highly enriched than other proteins that have since been reported in exosomes, we collected cell and exosome fractions from HEK293 cultures and processed them for immunoblot using antibodies specific for 24 different exosomal proteins.…”

Psoriasis to Psoriatic Arthritis: The Application of Proteomics Technologies

“…This review summarized the current approaches applied in the early PsA proteome. Compared with the traditional LC-MS/MS methods in proteogenomics, PEA provides more sensitive and specific detection for a more considerable range of low-abundant proteins in human blood and other body fluid samples ( 55 – 58 ). However, the need for the custom panel of biomarkers also restricted the exploration of the unknown proteins.…”

“…The unique DNA will be merged by high-throughput relative quantification microfluidic qPCR for up to 1,161 human proteins in the plasma ( 54 , 55 ). Compared with LC-MS/MS, PEA covers a broader dynamic range with higher sensitivity, which provides sensitive and specific detection of low-abundant proteins in human blood and other body fluid samples ( 55 – 58 ). Moreover, PEA also tends to be less influenced by multiplex ELISA technical problems, such as antibody crossreactivity and interassay variability ( 59 ).…”

Skin Cancer Research Goes Digital: Looking for Biomarkers within the Droplets

“…Except for the apoptotic bodies, all EVs are released into circulation by living cells, either by direct budding of the plasma membrane (oncosomes and microvesicles) or via the endosomal pathway (exosomes) [ 200 , 202 ]. EVs can be found in many biological fluids, including blood [ 203 ], amniotic fluid [ 204 ], urine [ 205 ], CSF [ 206 ], breast milk [ 207 ], saliva [ 208 ], ascites [ 209 ], tears [ 210 ], semen [ 211 ], bronchoalveolar lavage fluid [ 212 ], and in the conditioned media of cultured cells [ 213 ].…”