Exosomes are one of common extracellular vesicles (EVs) with diameter size of 30-150 nm. They are produced in endocytic compartments and then secreted to microenvironment, such as tissues and diverse biological fluids including serum/plasma, cerebrospinal fluid and urine (in vivo). Besides, exosomes can also be present in the conditioned media (in vitro). Definitely, the EVs have specialized and important functions in various physiological processes. However, compared with EVs in general, it is still unknow the unique characteristics or functions of exosome in clinical biomarker area. Therefore, with the develop of exosome isolation technique, there's a growing interest and make it possible to figure out the detailed functions or application of exosome in pathological process of diseases.
Exosomes mainly contain a mixture of proteins, lipids, RNAs, etc. According to current studies, the exosomes from different biological fluids have diverse contents and functions. Definitely, compared with other contents, proteins play a crucial role in biological functions. Therefore, to perform total protein profiling of exosomes from different biological samples are essential and useful in biomarker discovery and clinical diagnosis.
Creative Proteomics provides professional and comprehensive exosome proteomics analysis to identify and quantify the proteins in the exosomes. In addition, we can also help you with biomarker discovery after interpreting proteomics data by powerful bioinformatics technique.
Figure 1: workflow for saliva and serum exosome proteomics (Y. Sun et al., 2017)
We used three techniques, Label free, SILAC, and TMT/iTRAQ, to study the exosome quantitative proteome.
Label free method does not require isotope labeling reagents and is not limited by sample conditions, but accuracy is affected by factors such as mass spectrometry reproducibility.
SILAC technique is an in vivo labeling technique with stable labeling effect and 99% labeling efficiency, and is not affected by lysate composition.
TMT/iTRAQ is an in vitro labeling technique with high labeling sensitivity, good reproducibility and high coverage. And TMT labeling can compare up to 10 samples in parallel.
Creative Proteomics offers a variety of exosome isolation and extraction protocols depending on the characteristics of the sample. The main protocols include.
- Differential ultracentrifugation method.
Suitable for large volume samples such as cell supernatant, lavage fluid, urine, etc.
- Kits based on the principle of PEG precipitation.
Suitable for small volume samples such as serum, cerebrospinal fluid, etc.
- Ultrafiltration extraction method.
It is suitable for concentration of bacterial cultures or samples with low exosome. It can also be used with ultracentrifugation method to do further extraction.
- Qualitative and quantitative quality control
- Identification and differential protein statistics
- GO functional analysis
- KEGG pathway analysis
- KOG (COG) analysis
- Protein Interaction Networks
Advantage of Exosome Proteomics Service
- Comprehensive sample preparation. We can perform the whole project from exosome isolation to protein identification and quantification. Exosomes can be isolated from various samples including plasma/serum, cerebrospinal fluid, saliva, snot, urine, bile, cell media, etc.
- Cutting-edge facilities & optimized protocols. Our proteomics platform is equipped with Q Exactive HF MS and Orbitrap Fusion Lumos MS that are the newest version of MS with the highest resolution. Besides, our scientists are experienced in exosome proteomics area and can provide customized service.
- Biomarker discovery by bioinformatics analysis. Our bioinformaticians are experts in various bioinformatics tools for data interpretation and deeper mining.
With years' experience and state-of-the-art instrumentation, Creative Proteomics have a comprehensive workflow for exosome proteomics research. Please contact us for more detailed information.
How to isolate and extract exosomes?
There are several current methods of exosome isolation, and it is not possible to specify which isolation method is better; each of them has its own advantages and disadvantages. The separation method needs to be selected according to the characteristics of the sample.
- Ultracentrifugation method
The advantage is low cost, but the purity of the extracted exosomes is low and the recovery rate is unstable.
- Density gradient centrifugatio
The advantage is that the purity of the extracted exosomes is high, but the operation steps are tedious and time-consuming.
- Ultra-filtration centrifugation
Ultra-filtration centrifugation is simple and efficient, and does not affect the biological activity of exosomes, so it is a new method for extracting cellular exosomes.
- Magnetic immunoassay
The magnetic bead method has the advantages of high specificity, easy operation and does not affect the morphological integrity of exosomes, but the efficiency is low and the biological activity of exosomes is easily affected by pH and salt concentration.
- Polymer precipitation method
The advantages are simple operation and no additional equipment is needed, but the purity of isolated exosomes is low and they are easily contaminated by non-exosomal materials.
- Kit extraction
A variety of exosome extraction kits are available on the market, some of which filter out impurities through specially designed filters, while others use spatial exclusion chromatography for separation and purification.
How to identify the extracted exosomes?
Exosome identification is divided into three levels: transmission electron microscopy, Nanosight particle size, and protein markers. nta analyzes the particle size distribution and particle concentration of exosomes, TEM electron microscopy to observe the morphological characteristics of exosomes, and WB detects exosome proteins marker.
How to preserve exosomes?
If studying exosome morphological characteristics, proteins or other functions, it is best to use fresh exosomes after isolation.
The prevailing recommendation is that it is best to use fresh exosomes after extraction or to store them frozen. They can be kept at 4°C for a few days for short periods of time, and need to be stored frozen for longer periods of time.
How to determine the authenticity of exosomes?
- Artificial exosomes are not exosomes.
- Lyophilized exosome cultures are not exosomes.
- Exosomes may not have strong repair and regeneration ability if they are not derived from stem cells.
- Exosomes in liquid form at room temperature do not have biological functional activity and need to be frozen and preserved to maintain its activity.