Exosome Protein Profiling Service with Olink Proteomics Technology

Q: Q1: How does Olink's Proximity Extension Assay (PEA) work for exosome protein profiling?

Olink PEA combines antibody-based detection with nucleic acid amplification for ultra-sensitive exosome protein analysis. The process involves three key steps: first, pairs of oligonucleotide-conjugated antibodies bind to target proteins on exosomes, with one recognizing the extracellular domain and the other a nearby epitope. When both antibodies bind, their oligonucleotide tails are brought into proximity, triggering a DNA polymerase-mediated extension to form a unique amplicon. This amplicon is then quantified using real-time qPCR (Olink Flex) or next-generation sequencing (Olink Explore), with signal intensity directly proportional to target protein concentration. This dual-recognition mechanism eliminates cross-reactivity from free proteins or debris, ensuring high specificity for exosome-associated proteins.

Q: Q2: What sample volume is required for exosome protein profiling using Olink technology?

The required sample volume depends on the starting material: For exosome-enriched suspensions: Only 5-20 μL is needed, making it ideal for precious samples For raw biofluids: 50-200 μL of serum/plasma or 1-5 mL of cell culture supernatant Olink's technology can detect proteins from as little as 1 μL of plasma or other bodily fluids, demonstrating exceptional sensitivity for limited samples such as pediatric specimens or rare disease cohorts.

Q: Q3: How is exosome purity verified before Olink analysis?

We implement a three-tier validation system to ensure exosome purity: Nanoparticle Tracking Analysis (NTA) confirms size distribution (30-150nm) and concentration (≥10⁸ particles/mL) Western Blot detects exosome markers (CD63, CD9, TSG101) while excluding contaminants like albumin and histone H3 Transmission Electron Microscopy (TEM) verifies the characteristic cup-shaped morphology of exosomes For targeted enrichment, immunoprecipitation with anti-CD63/CD9/CD81 magnetic beads achieves ≥95% purity, while methods like size-exclusion chromatography effectively remove protein aggregates.

Q: Q4: What bioinformatics analyses are included in the service?

Our comprehensive data analysis pipeline includes: Basic analysis: Protein concentration matrix, CV calculation (<10% for technical replicates), and missing value imputation Differential analysis: Volcano plots, heatmaps, and fold-change analysis (FC ≥1.2, P < 0.05) Biological interpretation: GO/KEGG pathway enrichment and protein-protein interaction networks Clinical translation: ROC curve analysis with AUC calculation for biomarker validation Analyses are performed using industry-standard tools including MaxQuant, Perseus, and STRING, with results delivered as interactive visualizations and raw data files compatible with GraphPad Prism and R.

Q: Q5: Can I create custom panels for specific exosome proteins of interest?

Yes, custom panels are available for targeted exosome research. You can specify up to 384 proteins for inclusion in your panel, such as tissue-specific receptors, signaling molecules, or disease-associated biomarkers. Olink's technology supports detection of over 1,100 proteins across various panels, with the flexibility to combine markers from different pathways. Our scientific team will assist in panel design to ensure optimal antibody pairs and detection performance for your specific exosome research questions.

Q: Q6: What types of biological samples are compatible with this service?

Olink proteomics technology supports diverse sample types for exosome analysis: Biofluids: Serum, plasma, cerebrospinal fluid, and urine Cell culture: Supernatants from various cell types Tissue-derived: Exosome-enriched fractions from homogenized tissues The platform handles both fresh and frozen samples, with specific stabilization protocols recommended for each type. For example, serum/plasma samples should be stored at -80°C with protease inhibitors, while cell culture supernatants require prompt processing or preservation to maintain exosome integrity. All samples undergo rigorous quality control upon receipt to ensure suitability for analysis.

Due to the limited sample volume of EVs, detecting protein content requires technologies with high sensitivity and accuracy. Olink's PEA technology has gained popularity in EV research for its minimal sample consumption and exceptional detection sensitivity. Creative Biostructure's Olink proteomics service effectively addresses the limitations of conventional exosomal protein mass spectrometry by integrating high-purity exosome isolation with the proprietary Proximity Extension Assay (PEA) technology. Our combined approach enables ultrasensitive and highly specific detection of hundreds of exosomal proteins under extremely low sample input conditions, paving a new path for exosome research and clinical translation.

Introduction to Olink Technology and Its Application Value in Exosome Research

Exosomes carry specific proteins, nucleic acids, and other information from their source cells, making them a valuable source of biomarkers for disease diagnosis. However, due to their small size and limited contents, detecting low-abundance proteins has remained a technical challenge. The Olink PEA technology demonstrates significant advantages in this regard:

Unveiling the Exosomal Proteome

Olink's high sensitivity and multiplex detection capability allow researchers to deeply explore the protein composition of exosomes from limited clinical samples (such as small volumes of serum or plasma), enabling the discovery of characteristic protein expression profiles associated with diseases.

Tracing Cell Origins and Functions

By analyzing specific membrane proteins on the surface of exosomes (such as CD63, CD9, etc.) and their cargo proteins, Olink technology helps infer the cellular origin of exosomes and reveals their functions in processes like intercellular communication and immune regulation.

Discovery and Validation of Disease Biomarkers

The high sensitivity and stability of Olink technology make it well-suited for biomarker discovery and validation. Particularly in the field of inflammation research, Olink offers specialized panels that can efficiently capture a wide range of low-abundance cytokines. These cytokines play critical roles in immune and inflammatory responses but are difficult to effectively detect using traditional mass spectrometry methods.

Exosome Protein Profiling with Olink Proteomics Technology

Olink Proteomics technology, particularly its unique Proximity Extension Assay (PEA), provides a powerful tool for proteomic research of minimal samples such as exosomes. Our technology enables ultra-high sensitivity, high specificity, and highly multiplex protein detection and quantification, helping clients gain deeper insights into disease mechanisms, discover biomarkers, and advance the development of precision medicine.

Our exosomal protein analysis service, based on Olink Proteomics Technology, features the following characteristics:

Features	Description	Advantages
Ultra-high sensitivity	The detection sensitivity can reach the fg/mL (femtogram per milliliter) level.	Capable of effectively detecting low-abundance proteins (such as cytokines) in exosomes that are difficult to detect using traditional methods.
Minimal sample requirements	Only 1-6 μL of sample volume is required for testing.	Ideal for research involving precious or trace samples such as exosome lysates, cerebrospinal fluid, serum, and similar specimens.
High multiplexing capability	Simultaneously detects 48-3072 protein biomarkers.	Enable high-throughput screening to obtain extensive protein information in a single experiment, revealing complex protein regulatory networks.
Wide dynamic range	Dynamic range spanning 10 orders of magnitude.	Capable of simultaneously and accurately quantifying proteins at high, medium, and low abundances within samples, preventing signal saturation or loss.
High specificity and accuracy	By employing a dual-antibody pairing to recognize target proteins and integrating DNA barcoding technology, this approach effectively avoids antibody cross-reactivity and signal crosstalk issues commonly encountered in multiplex immunoassays.	Ensure the reliability and accuracy of test results while reducing the false positive rate.
Reproducibility	High technical redundancy.	Ensure data stability and reproducibility.
Compatible with multiple sample types	Suitable for various sample types including serum, plasma, cerebrospinal fluid, urine, cell culture supernatant, and exosome lysate.	Provides a unified, reliable proteomics analysis platform for samples from diverse sources, with particular expertise in detecting low-abundance proteins in bodily fluids.

How Exosome Protein Profiling Works with Olink Proteomics Technology?

Olink's PEA technology elegantly combines the specificity of immunoassays with the sensitivity of nucleic acid amplification. Its core workflow is illustrated in the figure below:

1

Antibody-Oligonucleotide Probes

Olink pairs of antibodies, each labeled with a unique oligonucleotide probe, bind specifically to target proteins on the exosome.

2

Proximity Event

When both antibody probes bind to the same target protein, the DNA oligonucleotides are brought into close proximity.

3

DNA Amplification

A connector DNA Oligonucleotide allows the linked probes to form a detectable DNA template that can be amplified via real-time PCR or NGS.

4

Signal Conversion

The amplified DNA signal is directly proportional to the amount of the target protein.

Workflow for exosomal protein analysis utilizing olink proteomics technology. Figure 1. Exosome Protein Profiling Workflow with Olink Proteomics Technology. (Creative Biostructure)

Data Analysis & Interpretation

We transform raw Olink data into actionable insights through multidimensional analysis:

Analysis Items	Analysis Content
Basic Analysis	Protein concentration matrix, inter-sample CV calculation (<10% for technical replicates), missing value imputation.
Differential Analysis	Volcano plots, heatmaps, and fold-change (FC) analysis to identify disease-associated exosome proteins.
Biological Interpretation	GO/KEGG pathway enrichment (e.g., "exosome-mediated signaling," "PI3K-AKT pathway"), protein-protein interaction (PPI) networks.
Clinical Translation	ROC curve analysis (AUC calculation) for biomarker validation, correlation with clinical outcomes (e.g., survival, treatment response).

Deliverables

QC report (exosome purity/morphology data, Olink assay quality metrics).
Raw data (Excel/FASTQ files) and processed data matrix.
Comprehensive analysis report with visualizations and biological interpretations.
Methodology document (for manuscript submission, compliant with Olink's technical standards).

Case Study

Case: Characterization of extracellular vesicles in metastatic urothelial cancer patients to discover protein signatures linked to therapeutic outcomes

Background

Patients with metastatic urothelial carcinoma (mUC) typically have a poor prognosis, making early prediction of systemic treatment response critical for improving patient outcomes. This study investigated the proteome of extracellular vesicles (EVs) isolated from consecutive plasma samples in a cohort of mUC patients receiving combination therapy with vinorelbine and sorafenib in a Phase I trial. The isolated EVs were of exosomal size and expressed exosomal markers CD9, TSG101, and SYND-1. The study found no association between the number of EVs per milliliter of plasma at baseline and progression-free survival (PFS). Proteomic analysis of the EVs using antibody-based adjacent extension analysis with the Olink Oncology II panel revealed heterogeneous protein expression patterns.

Key Findings

A protein signature composed of SYND-1, TNFSF13, FGF-BP1, TFPI-2, GZMH, ABL1, and ERBB3 is hypothesized to be associated with progression-free survival (PFS) in metastatic urothelial carcinoma (mUC).
Protein features associated with optimal treatment response were identified, including FR-alpha, TLR3, TRAIL, and FASLG.
Machine learning classification algorithms also identified several markers associated with PFS or optimal treatment response characteristics.

Association of extracellular vesicle protein analysis with progression-free survival (PFS). Figure 2. The relationship between extracellular vesicle protein analysis and progression-free survival (PFS). (Viktorsson K, et al., 2022)

Creative Biostructure is dedicated to providing clients with professional exosome protein analysis services. Our team of Olink-certified specialists and exosome researchers will provide tailored solutions to accelerate your research. Please contact us for a customized experimental design plan. We will be happy to assist you.

Reference

Viktorsson K, Hååg P, Shah C H, et al. Profiling of extracellular vesicles of metastatic urothelial cancer patients to discover protein signatures related to treatment outcome. Molecular oncology. 2022, 16(20): 3620-3641.

Frequently Asked Questions

For any inquiries, our support team is ready to help you get technical support for your research and maximize your experience with Creative Biostructure.

Related Services