Single-Vesicle Membrane Proteomics Services

Q: Q1: How does single-vesicle membrane proteomics achieve single-vesicle resolution? What are the core technologies?

Single-vesicle resolution relies on a dual technical system of "precision capture + microscale detection". Primary isolation uses microfluidic chip-based droplet trapping with 20-1000nm microchannels to physically separate vesicles by size and surface charge. For low-concentration samples, nanoparticle immunocapture with CD63/CD9 antibody-functionalized superparamagnetic beads ensures specific single-vesicle binding with <2% mixing rate. Captured vesicles undergo integrity verification via Atomic Force Microscopy (AFM) before analysis on an Orbitrap Exploris 480 mass spectrometer coupled with nano-LC system, enabling femtogram-level detection. Recent advancements like Droplet Barcode Sequencing (DBS-pro) further enhance throughput by compartmentalizing individual vesicles in emulsion droplets for barcoded protein profiling.

Q: Q2: How should vesicle samples be stored and transported? Will this affect results?

Samples must follow strict "cold-chain stabilization" protocols: Short-term storage (<72 hours): 4°C refrigeration without repeated freeze-thaw cycles Long-term storage: Aliquoted at -80°C with protease inhibitors (e.g., PMSF) to prevent protein degradation Transport requirements: Shipped on dry ice (maintaining ≤-70°C) within 72 hours with sample metadata sheet Studies show proper storage preserves >90% membrane protein integrity for 3 months, while improper handling increases degradation by 40%. We perform immediate quality control upon receipt and notify clients of any degradation issues.

Q: Q3: What sample size is required for clinical samples like tumor sera?

Follow these guidelines: - Exploratory studies: ≥10 samples/group for initial biomarker screening - Validation studies: ≥30 samples/group to ensure 95% reliability in ROC curve analysis (AUC validation) For rare disease samples, ≥5 samples/group are accepted with statistical limitations noted. We adjust strategies for small cohorts by increasing technical replicates (≥5) to mitigate individual variability.

Q: Q4: How do you confirm detected membrane proteins are vesicle-derived, not contaminants?

A three-tier validation system ensures specificity: Pre-isolation: NTA nanoparticle tracking confirms 20-1000nm particle size distribution. Post-isolation: Western Blot verifies vesicle markers (CD63/CD9) while excluding cell debris (histone H3) and serum proteins (albumin) with <5% contaminant signal. Mass spec validation: ≥80% of identified proteins annotated to membrane compartments via GO analysis, with no significant nuclear/cytoplasmic protein enrichment.

Q: Q5: What are the recommended technical and biological replicates? How is result reliability ensured?

We recommend: Technical replicates: ≥3 per sample with CV <15% between single vesicles Biological replicates: ≥5 animals/group or ≥30 clinical samples/group for statistical significance Reliability is guaranteed through: (1)≥98% vesicle purity verified by AFM and Western Blot for CD63/CD9 ; (2)≥95% peptide match rate using DDA mode with targeted validation ; (3)Traceable raw data with positive controls (e.g., Na+/K+-ATPase) ensuring <10% quantification bias.

Single-vesicle membrane proteomics service leverages cutting-edge technologies to conduct high-resolution, precise profiling of the protein landscape on the surface of individual extracellular vesicles. This groundbreaking approach serves as a pivotal tool for uncovering novel surface biomarkers, understanding intercellular communication mechanisms, and advancing targeted therapeutic development. Creative Biostructure provides state-of-the-art single-vesicle analysis that deciphers the functional membrane proteome with unparalleled detail, delivering critical insights for fundamental biomedical research and theranostic applications.

What are The Advantages of Single-Vesicle Membrane Proteomics?

Vesicles serve as core carriers for intracellular material transport and signal transduction. The composition and function of their membrane proteins, which directly determine cellular physiological and pathological states, are critical in diverse processes. These range from synaptic neurotransmission to the distal regulation of tumors mediated by exosomes. Therefore, deciphering the heterogeneity of vesicle membrane proteins is key to understanding the microscopic mechanisms of life.

Traditional bulk vesicle proteomics fails to capture membrane protein differences between individual vesicles due to averaging effects. In contrast, single-vesicle membrane proteomics services integrate a comprehensive technical system encompassing "single-vesicle precision separation - efficient membrane protein extraction - high-sensitivity mass spectrometry detection - multidimensional bioinformatics analysis," has pioneered the qualitative, quantitative, and functional analysis of membrane proteomes in individual vesicles. This revolutionary tool unlocks insights into vesicle heterogeneity mechanisms and facilitates the discovery of novel biomarkers and drug targets.

Our Single-Vesicle Membrane Proteomics Services

Creative Biostructure provides clients with a one-stop single-vesicle membrane proteomics solution, from sample preparation to bioinformatics analysis. Our services include but are not limited to the following:

Services	Description
Single-Vesicle Isolation and Enrichment	Tailor-made separation strategies for vesicles of different origins and sizes: Microfluidic Chip Isolation: Utilizing differences in vesicle size, density, and surface charge, this technique achieves precise isolation of individual vesicles through "droplet trapping" or "immunoaffinity capture" technologies on a microfluidic chip. Nano-Capture Magnetic Bead Technology: Utilizing superparamagnetic nanoparticles surface-modified with vesicle-specific antibodies (e.g., CD63, CD9, Synaptophysin) to efficiently enrich target single vesicles. Suitable for low-concentration samples (e.g., cerebrospinal fluid, serum-derived exosomes). Quality Control: Verification of single-vesicle integrity and purity via atomic force microscopy (AFM) or confocal laser scanning microscopy (CLSM).
Single-Vesicle Membrane Protein Extraction and Purification	To address the characteristics of membrane proteins, we employ an optimized extraction system: Mild Detergent Extraction: Select nonionic or zwitterionic detergents to efficiently separate membrane proteins from vesicle contents while maintaining membrane protein structural integrity. Descaling Agent Removal and Concentration: Remove descaling agents via centrifugal ultrafiltration tubes (3 kDa molecular weight cutoff) or the Sevag method to prevent interference with subsequent mass spectrometry analysis. Purity Verification: Signature membrane protein molecules are detected via SDS-PAGE gel electrophoresis or Western Blot.
High-Sensitivity Mass Spectrometry Detection	Qualitative Analysis: Utilize Data-Dependent Acquisition (DDA) mode to cover ≥500 membrane proteins within a single vesicle. Quantitative Analysis: Support label-free or TMTpro 16plex isotope-labeled quantification, enabling comparison of membrane protein expression differences in individual vesicles across different treatment groups (e.g., normal vs. tumor).
Proteomics Bioinformatics Analysis	We deliver multi-dimensional data analysis customized to our clients' specific needs: Basic Information Analysis: Protein identification list, quantitative result matrix, principal component analysis (PCA), and differential protein screening. Membrane Protein Characterization Analysis: Transmembrane Domain Prediction (TMHMM), Signal Peptide Prediction (SignalP), Membrane Protein Subcellular Localization (WoLF PSORT) Functional Notes: GO functional annotation, KEGG pathway enrichment analysis, reactome pathway analysis Interaction Network Analysis: Constructing membrane protein-protein interaction (PPI) networks based on the STRING database to identify core regulatory proteins (e.g., hub proteins) Customized Analysis

Workflow of Single-Vesicle Membrane Proteomics Analysis

We adhere to standardized and traceable experimental protocols to ensure reliable results at every step.

1

Sample Receipt and Evaluation

Accepted Sample Types: Fresh/frozen vesicle suspensions, cell lysates (vesicles must be pre-isolated by the client), bodily fluid samples (serum, cerebrospinal fluid, cell culture supernatants).
Sample Evaluation: Determine vesicle concentration (NTA nanoparticle tracking analysis), purity (Western Blot for contaminating proteins), and confirm experimental objectives with the client (e.g., qualitative/quantitative analysis, differential analysis direction).

2

Single-Vesicle Preparation and Quality Control

Select separation methods (microfluidics/magnetic bead capture) based on sample characteristics to isolate individual vesicles.
Verify single-vesicle integrity via AFM/CLSM and issue quality control reports.

3

Membrane Protein Extraction and Mass Spectrometry Analysis

Optimize the detergent system for membrane protein extraction, followed by concentration and nano-LC-MS/MS analysis.
Continuously monitor mass spectrometry data quality (e.g., peptide matching rate, protein coverage) to ensure data integrity.

4

Bioinformatics Analysis and Report Delivery

We perform multidimensional data analysis based on client requirements and generate visualization charts (e.g., volcano plots, heatmaps, pathway enrichment plots).
A comprehensive report is delivered, including: the experimental protocol, quality control data, raw mass spectrometry files (.raw), analysis result tables, figures, and biological interpretation of conclusions.

Service workflow for single-vesicle membrane proteomics analysis. Figure 1. Single-Vesicle Membrane Proteomics Analysis Workflow. (Creative Biostructure)

Application of Single-Vesicle Membrane Proteomics Analysis

Neuroscience

Elucidating the relationship between synaptic vesicle membrane protein heterogeneity and neurotransmitter release.

Investigating the sorting mechanism of amyloid precursor protein (APP) into single vesicles in Alzheimer's disease (AD).

Tumor Biology

Screening for tumor-derived exosomal single-vesicle membrane protein biomarkers (e.g., PD-L1, CD47) for early diagnosis.

Analyzing changes in single-vesicle membrane proteins of tumor cell exosomes after chemotherapy to predict drug resistance mechanisms.

Cell Biology

Investigating the sorting and trafficking of vesicle membrane proteins in the endolysosomal pathway.

Elucidating the impact of cellular stress (e.g., hypoxia, oxidative stress) on the composition of single-vesicle membrane proteins.

Drug Discovery

Identifying drug targets on single-vesicle membrane proteins (e.g., ion channels, transporters).

Evaluating the regulatory effects of drugs on single-vesicle membrane protein expression to optimize dosing regimens.

Cardiovascular Diseases

Analyzing the role of endothelial cell vesicle single-vesicle membrane proteins (e.g., eNOS) in vascular homeostasis.

Screening for single-vesicle membrane protein biomarkers in serum exosomes of myocardial infarction patients for prognostic evaluation.

Customer Support & Delivery

Client Requirements

Samples: Fresh/frozen vesicle samples (must specify sample type, processing method, and storage conditions);

Experimental Requirements: Clearly define analysis objectives (qualitative/quantitative, control group design, pathways or proteins of interest);

Special Requirements: Such as customized data analysis content, report formats, etc.

Deliverables

Raw experimental data: Mass spectrometry .raw files, SDS-PAGE/Western Blot raw images, AFM/CLSM quality control images.

Analysis report: Includes experimental methods, quality control results, protein identification/quantification lists, bioinformatics analysis charts and conclusions.

Supplementary materials: Technical methodology descriptions, software parameters used for data analysis.

Case Study

Case: Precise Trafficking of a Synaptic Vesicle Protein Subset Revealed by Single-Vesicle Proteomics.

Background

Protein sorting regulates neurotransmission by defining synaptic vesicle composition. While bulk studies have estimated average protein numbers, intervesicle variability and sorting precision remain unknown. To address this, we employed a single-molecule approach to quantify the abundance and variance of seven integral membrane proteins in brain synaptic vesicles.

Methods

Isolation of vesicles from brain homogenates
Preparation of vesicles for fluorescence imaging
Fabrication of micro-channels
Optical setup
Fluorescent imaging of single vesicles and single antibodies
Fluorescent puncta analysis
Data analysis and fitting

Conclusion

This study reports that four vesicular proteins (SV2, the proton ATPase, Vglut1, and synaptophysin 1) exhibit minimal intervesicular variation in quantity, indicating their high-precision sorting into vesicles. In contrast, the apparent numbers of VAMP2/synaptobrevin 2, synaptotagmin, and synapsin demonstrated substantial variability between vesicles. These findings impose constraints on protein-functional models of the synapse and suggest that differences in vesicular protein expression may influence vesicle composition and function.

Analysis of synaptic vesicle protein heterogeneity at the single-vesicle level. Figure 2. Quantification of polydispersed synaptic vesicle proteins. (Mutch S A, et al., 2011)

Ready to resolve vesicle heterogeneity one membrane at a time? Our single-vesicle membrane proteomics workflow combines precision isolation, high-sensitivity MS, and multidimensional bioinformatics to reveal actionable surface biomarkers and targets. Contact us to tailor the pipeline to your samples and get a fast, study-ready quotation.

Reference

Mutch S A, Kensel-Hammes P, Gadd J C, et al. Protein quantification at the single vesicle level reveals that a subset of synaptic vesicle proteins are trafficked with high precision. Journal of Neuroscience. 2011, 31(4): 1461-1470.

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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