Integrated Exosome Proteomics Service with Single-Cell and Tissue Analysis

Q: Q1: How is your integrated service better than doing single-cell, exosome, and spatial analysis separately with different providers?

The key advantage lies in seamless data linkage and sample efficiency, which separate services can't achieve: - Sample savings: We use the same tissue sample for all three analyses (e.g., a single FFPE block for LCM exosomes, spatial mIF, and FFPE-scRNA-seq), reducing waste by 60-80% vs. separate services that need 3+ aliquots. - Data consistency: Unified protocols prevent batch effects by using consistent methods, such as the same exosome isolation for both proteomics and spatial mapping, and enable direct tracing of cellular origins, exosome cargo, and tissue locations; in contrast, separate services often generate incompatible data formats. - Mechanistic depth: We integrate data to reveal communication loops (e.g., "tumor stem cells [single-cell data] secrete VEGF+ exosomes [exosome proteomics] that accumulate in stroma [spatial data]"), while separate services only provide isolated insights.

Q: Q2: For tumor microenvironment research, can this service identify which specific tumor cell subset secretes exosomes that suppress immune cells?

As a core application of our service, this objective is achieved through a typical workflow that includes: - Single-cell profiling: Use scRNA-seq/CyTOF to identify tumor cell subsets (e.g., cancer stem cells marked by CD44+CD24-) and immune-suppressive markers (e.g., PD-L1, TGF-β) in their gene/protein expression; - Exosome proteomics: Isolate exosomes from the same tumor tissue (via LCM of tumor regions) and detect immune-suppressive proteins (e.g., PD-L1+ exosomes) using Orbitrap mass spectrometry; - Spatial mapping: Use mIF to co-localize PD-L1+ exosomes with CD3+ T cells (immune targets) in tumor stroma; - Integration: Match CD44+CD24- tumor cells (single-cell data) to PD-L1+ exosomes (proteomics) and their T cell-suppressive locations (spatial data), confirming the subset's role in immune suppression.

Q: Q3: If I've already isolated exosomes from my tissue samples, can I use them for the integrated service (instead of sending raw tissue)?

Yes. we accept pre-isolated exosomes, but they must meet our QC criteria to ensure compatibility: - Concentration: ≥1×10⁸ particles/mL (measured via NTA); - Purity: No detectable cell debris (TEM) or contaminating proteins (Western Blot for histone H3/albumin <5%); - Storage: Frozen at -80°C without repeated freeze-thaw cycles, and shipped on dry ice. If you provide pre-isolated exosomes, we'll skip the tissue exosome isolation step and integrate your exosomes with single-cell/spatial data from the same tissue (you'll still need to send raw tissue for single-cell/spatial analysis). This can shorten the service timeline by 1-2 weeks.

The high heterogeneity of cells and tissues presents a major bottleneck that hinders researchers from deeply deciphering the functional mechanisms of exosomes and discovering highly specific disease biomarkers. Creative Biostructure has innovatively introduced an integrated solution combining exosome proteomics, single-cell analysis, and tissue analysis. Our strategy effectively addresses the heterogeneity challenges in exosome research, significantly mitigating study biases caused by sample variability. Our specialized services provide robust evidence for cell tracing of exosomes and their regulatory mechanisms in disease, accelerating the discovery of clinically relevant biomarkers for our clients.

Why Integrate Exosomal Proteomics with Single-Cell and Tissue Analysis?

Exosomes are a type of extracellular vesicles, ranging in size from 50 to 150 nm, which carry various components (including nucleic acids, proteins, lipids, metabolites, and cell surface proteins) from their parent cells and shuttle through intercellular spaces to facilitate signal transmission in cell-to-cell communication. Since nearly all cell types secrete exosomes, those isolated from conventional research samples exhibit significant heterogeneity, including variations in size, cargo, function, and cellular origin.

Integrating exosome proteomics, single-cell omics, and tissue analysis technologies, this approach aims to explore the relationship between individual cells and their secreted exosomes within the tissue microenvironment. This cutting-edge service enables researchers to trace exosomes back to their specific cellular origins, decipher their protein cargo, and map their distribution and interactions in situ. This integrated analytical approach reveals cellular heterogeneity, exosomal communication mechanisms, and potential disease biomarkers with high resolution and multidimensional insight, providing robust support for precision medicine and drug development.

Our Integrated Exosome Proteomics Service with Single-Cell and Tissue Analysis

Creative Biostructure has developed a specialized single-cell combined exosome research service through extensive technical R&D. Clients provide a single tissue sample, which undergoes specialized composite collagenase digestion to separate tissue into single cells and interstitial fluid. Single-cell sequencing (scRNA-seq) of these cells yields cell subpopulation classification information within the tissue. Subsequently, proteomic analysis of exosomes in the interstitial fluid reveals the classification of tissue-derived exosome subpopulations. Integrating single-cell sequencing data with the exosome omics data enables identification of primary donor cells secreting tissue-derived exosomes or primary recipient cells internalizing them. This provides a crucial foundation for in-depth mechanistic studies and biomarker tracing.

Workflow of Integrated Exosome Proteomics Service with Single-Cell and Tissue Analysis

Our integrated service provides a seamless, end-to-end workflow designed to unlock a deeper understanding of cellular communication within the native tissue microenvironment. We guide your project from a single tissue sample to biologically actionable insights through a meticulously coordinated process.

1

Sample Consultation & Design

Receive sample types: Fresh/frozen tissue sections (10-20 μm), dissociated tissue cells, or tissue-derived exosome suspensions.
Collaborate with clients to define research goals (e.g., "Identify exosome sources in breast cancer tissue") and select modules (e.g., scRNA-seq + exosome proteomics + mIF).

2

Sample Processing & Multi-Omics Detection

Tissue preparation: FFPE or fresh frozen tissue sectioning, LCM for region-specific isolation.
Parallel analyses
Single-cell omics: scRNA-seq/CyTOF of dissociated tissue cells.
Exosome analysis: Isolation + MS-based proteomics of tissue-region exosomes.
Spatial omics: Visium spatial transcriptomics or mIF of tissue sections.

3

Data Integration & Analysis

Use custom bioinformatics pipelines (R, Python, Seurat, SpatialData) to unify multi-omics data.
Generate visualizations (spatial heatmaps, interaction networks, volcano plots) and statistical validation (P < 0.05, FC ≥ 1.2).

4

Report Delivery & Post-Service Support

Deliver comprehensive report: Experimental methods, QC data (e.g., NTA/TEM images, scRNA-seq cell clustering), integrated analysis results, and biological conclusions.
Provide 1:1 data interpretation meetings with multi-omics experts.
Assist with manuscript preparation (methodology sections, figure legends).

A Unified Workflow for Integrated Exosome, Single-Cell, and Tissue Analysis. Figure 1. Workflow of integrated exosome proteomics service with single-cell and tissue analysis. (Creative Biostructure)

Deliverables

Raw data: scRNA-seq FASTQ files, exosome MS .raw files, spatial omics count matrices.
Processed data: Cell clustering results, exosome protein lists (with quantification), spatial maps.
Analysis report: Integrated conclusions, statistical tables, and high-resolution visualizations.
Technical documentation: Sample processing protocols, QC certificates, and software parameters.

Case Study

Case: Multi-omics Profiling of Serum EVs Identifies MACROH2A1 in Refractory COVID-19.

Background

To identify potential serum biomarkers and examine proteins associated with the pathogenesis of refractory COVID-19, this study performed high-coverage proteomic analysis on serum extracellular vesicles collected from 12 COVID-19 patients with varying disease severity and 4 healthy controls. Additionally, single-cell RNA sequencing was conducted on peripheral blood mononuclear cells (PBMCs) obtained from 10 COVID-19 patients and 5 healthy controls.

Methods

Isolation of EVs for proteomics.
Proteomic analysis.
Bioinformatics analysis of the proteome.
Analysis of molecular networks and pathways for the proteome and transcriptome.
Cell culture and stimulation.
Transmission electron microscopy.
Subjects and specimen collection of PBMCs for scRNA-seq.
Droplet-based single-cell sequencing.
Alignment, quantification, and quality control of scRNA-seq data

Conclusion

The study demonstrates that MACROH2A1 in extracellular vesicles serves as a potential biomarker for refractory COVID-19, possibly reflecting the disease pathogenesis in monocytes.

$Multi-omics integration of serum EV proteomics and scRNA-seq identifies MACROH2A1 in refractory COVID-19.$ Figure 2. Next-generation proteomics of serum extracellular vesicles combined with single-cell RNA sequencing identifies MACROH2A1 associated with refractory COVID-19. (Kawasaki T, et al., 2022)

Ready to turn tissue heterogeneity from noise into insight? Our integrated exosome proteomics, single-cell, and tissue analysis workflow traces vesicles back to their cellular origins, maps their cargo in situ, and links them to clinically relevant phenotypes with multi-omics, publication-ready reports. Contact us to tailor your study design and receive a fast, expert quotation.

Reference

Kawasaki T, Takeda Y, Edahiro R, et al. Next-generation proteomics of serum extracellular vesicles combined with single-cell RNA sequencing identifies MACROH2A1 associated with refractory COVID-19. Inflammation and Regeneration. 2022, 42(1): 53.

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.

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