Coupling size exclusion chromatography to ultracentrifugation improves detection of exosomal proteins from human plasma by LC-MS.
Author(s): Alameldin S, Costina V, Abdel-Baset HA, Nitschke K, Nuhn P, Neumaier M, Hedtke M
Publication: Pract Lab Med, 2021, Vol. 26, Page e00241
PubMed ID: 34258353 PubMed Review Paper? No
Purpose of Paper
This paper investigated if size-exclusion chromatography (SEC) and ultracentrifugation can be used to enrich exosomes isolated from the plasma of healthy volunteers.
Conclusion of Paper
While mean protein concentration was much higher when exosomes were isolated by ultracentrifugation alone when compared to ultracentrifugation followed by SEC (288 µg/mL versus 26.4 µg/mL), a larger percentage of the 100 most frequently identified human proteins in the database ExoCarta was observed when ultracentrifugation was combined with SEC than when ultracentrifugation was used alone. Further, use of SEC after ultracentrifugation decreased plasma protein contamination, including a 95% reduction in levels of the three albumin peptides investigated.
Studies
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Study Purpose
This study investigated if size-exclusion chromatography (SEC) can be used with ultracentrifugation to enrich for exosomes in plasma collected from healthy volunteers. EDTA blood was obtained from four healthy volunteers and plasma was separated within 30 min by centrifugation without braking at 1600 g for 10 min followed by 3000 g for 10 min. When possible, fresh plasma was used for exosome isolation, but in some cases plasma was frozen at -20°C for an unspecified duration. Vesicles were isolated from plasma by centrifugation at 2000 g for 10 min followed by10,000 g for 20 min to eliminate cells.The supernatant was then diluted in phosphate buffered saline and macrovesicles were removed by centrifugation at 14000 g for 30 min. Exosomes were then pelleted by centrifugation at 100,000 g for 1.5 h, washed in PBS, centrifuged again at 100,000 g for 1.5 h, and resuspended in PBS. To further purify exosomes, three or twelve fractions were collected from the mini-PURE EVs SEC mini Columns. The isolated fractions were concentrated using a speed vacuum concentrator. Total protein was quantified by Qubit. CD9 and CD63 expression were quantified by Western Blot. Particle size distribution and count were assessed by nanoparticle tracking analysis. Proteins were identified by LC-MS/MS, and those identified were compared to the 100 ExoCarta list of 100 human proteins mostly identified in exosomes.
Summary of Findings:
The highest levels of CD9 and CD63 and the greatest number of particles of the targeted diameter (94-113 nm) were isolated from SEC fraction 2 (when 3 fractions were collected) and from fractions 5-8 when 12 fractions were collected; subsequent analysis was limited to these fractions. Mean protein concentration was much higher when exosomes were isolated by ultracentrifugation alone as opposed to ultracentrifugation followed by SEC (288 µg/mL versus 26.4 µg/mL). While only 9-14 of the 100 human proteins mostly commonly identified exosomes were observed in exosomes isolated by ultracentrifugation, 34-43 proteins were observed among exosomes isolated when ultracentrifugation was combined with SEC. Further, use of SEC after ultracentrifugation decreased plasma protein contamination, including a 95% reduction in levels of the three albumin peptides investigated.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Fluorometry Protein LC-MS or LC-MS/MS Protein Western blot Morphology Light scattering Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Western blot Specific Targeted peptide/protein CD9
CD63
Analyte Extraction and Purification Analyte isolation method Ultracentrifugation
Ultracentrifugation and SEC