A Comparative Proteomics Study of Six Serum Exosome Isolation Procedures

Significance of the Topic

Exosomes are extracellular vesicles (30–150 nm) that carry proteins, nucleic acids and lipids, and they represent a promising source of biomarkers for diagnostics and liquid biopsy applications. Reliable proteomic profiling of serum-derived exosomes requires isolation methods that balance yield and purity to ensure detection of low-abundance vesicle proteins while minimizing co-isolated serum contaminants such as albumin.

Objectives and Study Overview

The study aimed to perform a head-to-head comparison of six serum exosome isolation strategies for downstream mass spectrometry-based proteomics and western blot validation. The six methods included:

• Differential ultracentrifugation with sucrose cushion (UC_S)
• Total Exosome Isolation Reagent for serum (TEI)
• qEV iZON size-exclusion chromatography
• MagCapture PS affinity capture
• ExoEasy Maxi spin column
• ExoSpin polymer-based precipitation

Performance metrics were total protein yield, proteome coverage, enrichment of known exosome markers (CD9, CD63, CD81, Alix, HSPA8, ANXA5, ICAM1, EPCAM, FLOT1, HSPA1A) and depletion of serum albumin (HSA).

Methodology and Instrumentation

Exosomes were isolated from pooled normal human serum by each method. Samples were lysed either in RIPA buffer (for western blot) or 5 % SDS (for proteomics). Proteins were buffer-exchanged into 0.1 % SDS/50 mM TEAB, reduced, alkylated and digested with MS-grade trypsin using a rapid in-solution protocol. SDS was removed by SCX cleanup. Peptides were enriched and desalted on an Acclaim PepMap 100 C18 trap and analyzed by nanoLC-MS/MS on a Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLCnano system coupled to a Thermo Scientific™ Q Exactive™ Plus Orbitrap. Data were processed in Proteome Discoverer and matched against the ExoCarta database to evaluate purity. Parallel western blots monitored CD9, CD63, CD81 and HSA.

Main Results and Discussion

Protein yield per mL of serum varied widely: TEI reagent yielded the highest protein amount (~5.2–8.3 mg/mL), while MagCapture recovered the lowest. Proteome analysis identified 620–700 protein groups from qEV iZON, MagCapture, UC_S and ExoEasy Maxi, compared to fewer identifications for ExoSpin and TEI. Key findings included:

• MagCapture provided the highest exosome purity, covering ~88 % of ExoCarta Top103 markers and depleting abundant serum proteins.
• qEV iZON and UC_S also yielded relatively pure vesicles but with slightly lower marker coverage.
• ExoSpin and TEI co-isolated high levels of serum albumin, suppressing detection of lower-abundance exosome proteins.
• Differential ultracentrifugation with sucrose cushion (UC_S) improved purity over simple UC but did not match MagCapture performance.

Benefits and Practical Applications

High-purity exosome preparations enable deeper proteome coverage, facilitating biomarker discovery and quantitative proteomics. The MagCapture kit emerges as the preferred method for proteomic studies where vesicle purity is critical. For workflows that require higher yield but tolerate lower purity, size-exclusion (qEV iZON) or precipitation-based methods may be acceptable. Density gradient ultracentrifugation remains an alternative when commercial kits are not feasible.

Future Trends and Opportunities

Advances in exosome isolation are expected to focus on microfluidic and affinity-based platforms for rapid, high-throughput processing. Integration with multi-omic analyses (proteomics, transcriptomics, lipidomics) and standardized protocols will improve reproducibility. Novel ligands and surface markers may allow selective capture of specific exosome subpopulations, enhancing diagnostic and therapeutic applications.

Conclusion

This comparative study demonstrates that the choice of isolation method strongly influences exosome proteome quality. MagCapture affinity capture delivers the highest purity at the expense of yield, while TEI precipitation offers maximum protein recovery but poor selectivity. Researchers should select isolation strategies aligned with their downstream analytical priorities, balancing yield, purity and throughput.

Used Instrumentation

• Thermo Scientific™ Q Exactive™ Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer
• Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLCnano System
• Acclaim™ PepMap 100 C18 Trap Column
• Pierce™ Protein Concentrators PES (10 kDa MWCO)
• High-speed ultracentrifuge with sucrose cushion capability

References

1. Kowal J, et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. PNAS. 2016;113(8):E968–E977.
2. Keerthikumar S, et al. ExoCarta: a web-based compendium of exosomal cargo. Journal of Molecular Biology. 2016;428(4):688–692.
3. Oliveros JC. VENNY. An interactive tool for comparing lists with Venn Diagrams. 2007.