How PROSPR Is Unlocking Neuroscience's Tiny Messengers
Imagine your brain's cells constantly sending tiny, sealed packages to one another—these packages contain vital instructions that maintain healthy brain function, repair damage, and coordinate complex activities.
These microscopic parcels, known as extracellular vesicles (EVs), represent one of neuroscience's most exciting frontiers. For decades, scientists struggled to isolate these elusive messengers from the complex tissues of the brain. That all changed with the development of an ingenious method called PROSPR (Protein Organic Solvent Precipitation), which has finally allowed researchers to unpack the brain's secret postal system and read its long-hidden messages 1 .
Diameter range of extracellular vesicles
Of CNS cells release EVs
Success rate with PROSPR method
Extracellular vesicles are often described as the body's "cellular postal system"—membrane-wrapped packages that carry molecular cargo between cells. In the brain, this communication network is particularly vital, allowing nerve cells to coordinate everything from learning and memory to damage responses 1 .
Why has studying brain-derived EVs been so challenging? The central nervous system contains only trace levels of EVs amidst a complex background of soluble proteins, protein aggregates, and proteolytic contaminants 1 .
PROSPR (Protein Organic Solvent Precipitation) represents a revolutionary approach to EV isolation that turns traditional methods on its head. Instead of trying to pellet the delicate vesicles themselves, PROSPR takes the opposite tack—it precipitates and removes soluble proteins using cold organic solvents, leaving the lipid-encapsulated EVs behind in suspension 3 .
Brain tissue is gently homogenized to release EVs without destroying their structural integrity.
A cold organic solvent (typically acetone) is added to the sample in a 4:1 ratio, causing soluble proteins to precipitate.
In the groundbreaking 2016 study that introduced PROSPR for CNS tissues, researchers designed a comprehensive experimental workflow to validate the method's effectiveness 1 .
The analysis revealed that PROSPR significantly outperformed ultracentrifugation in several key areas:
| Category | Findings | Significance |
|---|---|---|
| Total Proteins Identified | 2,901 proteins | Comprehensive profiling capability |
| Known EV Markers | 86 of top 100 exosomal markers | High confidence in genuine EV isolation |
| Novel Identifications | 685 proteins | Expands known human EV-associated proteome |
| Membrane Proteins | 42.43% of total | Consistent with expected EV composition (≥30%) |
| Comparison to Ultracentrifugation | 7.2-fold more vesicle-associated proteins | Substantial improvement over traditional method 1 |
| Lipid Class | Examples Identified | Status in EV Research |
|---|---|---|
| Phosphatidylserines (PS) | PS36:2, PS40:4, PS40:6 | Hallmark EV lipid family |
| Sphingomyelins (SM) | Multiple isoforms detected | Characteristic component of EVs |
| Lysophosphatidylcholines (lysoPC) | Several novel isoforms | Previously reported in EVs |
| Lysophosphatidylethanolamine (lysoPE) | Newly identified forms | Known EV component |
| Phosphatidylglycerol (PG) | Novel identifications | Expanded understanding of EV lipids 1 |
| Parameter | PROSPR | Ultracentrifugation |
|---|---|---|
| Sample Required | ~150 mg brain tissue | Larger samples typically needed |
| Processing Time | Hours | Typically 1-2 days |
| Protein Identification | 2,901 proteins | Significantly fewer |
| Contaminating Proteins | Dramatically reduced albumin | High levels of co-isolated proteins |
| Match to EV Databases | 90.7% match with Vesiclepedia | 78.0% match with Vesiclepedia |
| Practical Accessibility | Inexpensive, standard equipment | Requires specialized ultracentrifuges 1 3 |
| Reagent/Equipment | Function in PROSPR Protocol | Technical Notes |
|---|---|---|
| Cold Acetone | Precipitates soluble proteins while leaving EVs in suspension | Optimal solvent determined through comparative testing |
| Lysis Buffer (≥5% SDS) | Disrupts EV membranes to release cargo for analysis | Higher SDS concentrations improve protein detection |
| Protease Inhibitors | Prevents degradation of protein cargo during processing | Essential for maintaining sample integrity |
| LC-MS/MS System | Identifies and quantifies protein content | Enables high-throughput proteomic profiling |
| ESI-MS/MS | Characterizes lipid composition | Allows comprehensive lipidomic analysis |
| Flow Cytometer | Determines vesicle size distribution and concentration | Confirms presence of appropriately sized particles 3 |
Optimal solvent determined through systematic comparisons with alternatives like chloroform and trichloroacetic acid 3
Concentrations ≥5% required for efficient EV lysis, determined through careful optimization experiments 3
Doesn't require specialized equipment beyond standard molecular biology laboratory tools 1
The development of PROSPR for CNS-derived EVs opens numerous exciting avenues for neuroscience research and potential clinical applications. The method's ability to efficiently isolate EVs from small tissue samples makes it particularly valuable for studying human brain disorders where sample availability is often limited.
The discovery of 685 novel EV-associated proteins and numerous new lipid isoforms through PROSPR-based analysis 1 dramatically expands the landscape of potential biomarkers for neurological conditions.
Looking ahead, PROSPR may accelerate the development of EV-based therapeutics. Researchers are already exploring how EVs might be harnessed to deliver drugs across the challenging blood-brain barrier 2 .
The ability to efficiently isolate and characterize CNS-derived EVs could prove invaluable in therapeutic development.
The PROSPR method represents more than just a technical improvement—it's a key that unlocks a long-inaccessible world of cellular communication in the brain. By providing a simple, efficient, and accessible way to isolate extracellular vesicles from central nervous system tissues, PROSPR has democratized the study of these crucial messengers.
As researchers worldwide adopt this method, we can anticipate accelerated discoveries in fundamental neurobiology, as well as new diagnostic and therapeutic approaches for the most challenging neurological disorders.
References to be added manually in this section