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Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation.

Author(s): Askeland A, Borup A, Østergaard O, Olsen JV, Lund SM, Christiansen G, Kristensen SR, Heegaard NHH, Pedersen S

Publication: Biomedicines, 2020, Vol. 8, Page

PubMed ID: 32722497 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the effects of EV isolation method on the size, abundance, protein expression, and contamination levels in plasma EVs from healthy individuals. Blood was collected after an overnight fast from two healthy males and one female using a 21-gauge needle into sodium citrate vacutainers. After discarding the first tube, platelet poor plasma was obtained by dual centrifugation at 2500 x g for 15 min at room temperature. Aliquots of PPP were snap-frozen in liquid nitrogen and stored at -80°C within two hours of blood collection. PPP was thawed in ice water for one hour before EV isolation by high-speed centrifugation, SEC, or PAP. High-speed centrifugation included five cycles of centrifugation at 18,890 x g for 30 min at room temperature and washing in phosphate-buffered saline (PBS)-citrate buffer. Size exclusion chromatography was performed using the qEV Original SEC columns and ultrafiltration using 100 kDa MWCO spin filters. Peptide affinity precipitation was performed using the New England Peptide ME Kit. Isolation of EVs and lack of lipoprotein contamination was confirmed by Western blotting using antibodies against CD9 and apolipoprotein B, respectively. Particle size and concentration were assessed by nanoparticle tracking using a NanoSight LM10-HS. Particles were further characterized and CD9 expression confirmed by transmission electron microscopy. Protein and peptide expression were evaluated by liquid chromatography-tandem mass spectrometry.

   Summary of Findings:

   All three methods isolated CD9 positive extracellular vesicles. Isolation by high-speed centrifugation resulted in larger particles than by SEC or PAP (mode 153 nm ± 6 nm versus 80 nm ± 7 nm and 91 nm ± 5 nm, respectively). The concentration of particles was also lower when isolation was by high-speed centrifugation than by SEC or PAP (3.69x 10⁹ versus 6.40 x 10¹⁰ and 6.81 x 10¹¹, respectively). Contrary to nanoparticle tracking analysis, relative intensity-based absolute quantification values (riBAQ) found 5.5-fold higher EV levels when isolated using high-speed centrifugation rather than SEV (P<0.001) and 19-fold higher EV levels compared to PAP (P=0.005). EVs isolated by high-speed centrifugation had the most proteins identified (808 versus 765 by SEC and 569 by PAP); however, 509 of the proteins were found regardless of isolation method. A further 235 were common between SEC and high-speed centrifugation, 36 between PAP and high-speed centrifugation, and 11 between SEC and PAP. The number of EV-specific proteins isolated was 21 when isolated by high-speed centrifugation, 22 by SEC, and 17 by PAP. Hierarchical clustering based on the protein profile clustered specimens by isolation method rather than by patient. Further analysis showed that high-speed centrifugation isolates had significantly more proteins associated with large EVs than SEC or PAP isolates (P<0.01, both) and significantly fewer proteins associated with small EVs than SEC or PAP isolates (P<0.01 and P<0.05, respectively). SEC isolates had more proteins associated with small EVs than PAP isolates (P<0.01) but a comparable number of proteins associated with large EVs.

   Serum albumin contamination was higher in isolated EVs obtained by high-speed centrifugation than by SEC (7.6-fold, P=0.003) or PAP (5.4-fold, P=0.008). In contrast, lipoprotein contamination was highest when isolation was with SEC rather than high-speed centrifugation (3-fold, P=0.001) or PAP (4-fold, P=0.003). The higher levels of lipoprotein contamination in SEC isolates was verified by Western blot analysis. Levels of non-EV associated proteins in PAP isolates were 19.8-fold higher than in SEC isolates (P=0.02) and 12.5 -fold higher than in high-speed centrifugation isolates (P=0.01). The reproducibility of the protein abundances was highest when extraction was with PAP (90%) followed by high-speed centrifugation (87%) and SEC (79%). While there was no similarity in overall protein abundances among the three methods, when only EV specific proteins were considered, there was similarity between isolates produced by high-speed centrifugation and SEC (R²=0.27-0.81).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Morphology	Light scattering
   Morphology	Electron microscopy
   Protein	Western blot
   Protein	LC-MS or LC-MS/MS
   Peptide	LC-MS or LC-MS/MS
   Lipoprotein	LC-MS or LC-MS/MS
   Lipoprotein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	High speed centrifugation Size exclusion chromatography Peptide affinity precipitation
   LC-MS or LC-MS/MS Specific	Technology platform	NTA

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