Application of Proteonano™ Platform for Plasma EV Proteomics Analysis

Extracellular vesicles (EVs) are bioactive cell-secreted nanoparticles that carry nucleic acids, proteins, lipids, and metabolites. They are involved in various physiological and pathological processes as key mediators of intercellular communication. EVs have important research value and application potential in exploring disease mechanisms, and developing clinical diagnostics and new drugs. As nanoscale biomarkers in liquid biopsy, EVs have shown great promise in clinical diagnosis. EVs can also be engineered as effective autologous vectors for precision drug delivery. However, the heterogeneity in the origin and molecular composition of vesicles secreted by cells poses challenges for the study and application of EVs.

The enrichment of EVs is the key to study their biological function and proteomics. The commonly used methods include ultracentrifugation, PEG precipitation, immunoaffinity, and TiO2 enrichment. Ultracentrifugation is commonly used for the enrichment of large volumes of samples lacking complexity. The magnetic nanoparticle-based EV enrichment method can greatly reduce the sample size requirement, and the extracted EV proteins can be used for high-throughput proteomics studies. At present, traditional EV enrichment methods based on magnetic bead technology have limitations such as incomplete enrichment, data gaps, inconsistent preprocessing, and lack of standardization, which lead to low coverage and poor reproducibility of protein data and affect the accuracy and reliability of data.

Amerigo Scientific offers the Proteonano™ platform as a powerful and reliable solution for plasma EV proteomics. Its robust performance and strong specificity make it a powerful choice for proteomics analysis of a variety of biological samples, contributing to advancements in proteomics research.

Key Instrument and Software

The performance of the Proteonano™ Ultraplex Proteomics Platform for plasma EV proteomics analysis was evaluated. The plasma sample used in the study was 40 μL with a loading amount of 200 ng. The high-resolution LC-MS/MS instrument chosen to be used was Thermo Scientific Orbitrap Astral with Vanquish Neo UHPLC.

Proteonano™ Platform

Plasma EV Protein Enrichment Efficiency

Proteonano™ EV Proteome Kit was used to enrich EV proteins in healthy human plasma. Transmission electron microscopy (TEM) imaging showed that the Proteonano™ EV kit had a significant enrichment effect on EV proteins. The magnetic beads in the Proteonano™ kit were coated with exosome particles that are uniform in size and intact in shape (approximately 30-150 nm in diameter). The background contained minimal impurities, indicating that the use of the Proteonano™ EV kit effectively reduced interference from non-target proteins and minimized non-specific binding during the isolation process.

Figure 1. TEM image of plasma EV proteins enriched using the Proteonano™ EV Proteome Kit.

Protein Identification and Dynamic Range

The Proteonano™ EV Proteome Kit and Nanomation™ G1 high-throughput sample preparation system enabled deep protein coverage with ng-level protein samples. Mass spectrometry was performed using the next-generation Orbitrap Astral high-resolution spectrometer with the Vanquish NEO liquid chromatography system. Peptides were dissolved in buffer and injected via the auto-injector, with 200 ng of peptides loaded for separation. An 8-minute gradient was used, and data was acquired in DIA mode. The results showed that about 3,040 EV proteins were identified in the groups using Proteonano™ Ultraplex Proteomics Platform, compared to about 2,400 proteins identified in the groups using standard magnetic beads (MS) and commercial reference beads (YWMS).

Figure 2. Number of EV proteins identified using the Proteonano™ EV Proteome Kit

Coverage of EV Proteins in Databases

The EV protein data from the samles treated with Proteonano™ EV Proteome Kit and with the commercial kits, respectively, were compared with the 5,181 proteins listed in the ExoCarta database. The protein data from the Proteonano™ kit covered up to 31.2% of the database proteins, while protein data from the commercial kit covered around 23%. The results showed the strong specificity of the Proteonano™ EV Proteome Kit for enriching plasma EV proteins. A comparison of EV proteins enriched using the Proteonano ™ kit with the Top 100 EV proteins in the ExoCarta database showed that the Proteonano™ kit enriched more of the top 100 proteins than commercial kits.

In addition, the EV proteins enriched by the Proteonano™ kit was analyzed and compared with Vesicode@Surface550 EV protein data, and it was found that Proteonano™ kit enriched about 20 more Surface550 proteins than the commercially available kit.

Figure 5. Overlap with Vesicode@Surface550 EV Proteins

Advantages of Proteonano™ Platform for Plasma EV proteomics

The Proteonano™ platform is an ideal sample preparation solution for EV proteomic analysis, which can be used in many fields such as disease research, diagnostic development, molecular mechanism research, and therapeutics discovery.

• Superior Analytical Performance: The Proteonano™ platform outperforms commercial kits in plasma EV proteomics, enriching more EV proteins with higher sensitivity, as demonstrated by comparisons with ExoCarta Top 100 and Surface550 datasets.
• Efficient Target Protein Capture: Based on the optimized exosome isolation technology, Proteonano™ platform efficiently extracts EVs from plasma while minimizing loss and reducing non-target protein interference. This platform is a highly specific and reliable EV enrichment tool.
• High Reproducibility and Standardization: The platform integrates automation and rigorous data analysis workflows, ensuring consistent and reproducible results.