I am currently preparing research methods for my thesis. My sample is an exosome from Adipose. However, I had difficulty identifying what types of proteins were in it. Can anyone help me? Thank You
Hello Assyfa Atha
Protein cargos are heterogeneous even between exosomes of the same type, but some proteins are consistently found in specific exosome types due to the roles of these proteins in exosome biogenesis and protein sorting.
Exosomes, because of their endosomal origin, contain protein families associated with the formation of intraluminal vesicles (ILVs) namely,Tsg101, tetraspanins, and Alix, membrane transport and fusion proteins namely, annexins, GTPases and flotillins, heat shock proteins namely, Hsc70, Hsp90, major histocompatibility complex (MHC) proteins namely, MHC I and MHC II, and cytoskeleton proteins namely, actin, myosin, and tubulin, which are usually regarded as markers to identify exosomes . In addition to proteins, you will find exosomes enriched in lipids such as cholesterol, glycosphingolipid, phosphatidylserine, and ceramide, which are essential for maintenance of exosome morphology.
The exosomal cargoes are normally recruited during ILV formation, in which, membranes of early endosomes partly invaginate and reverse bud into surrounding lumina with cytoplasmic content to form ILVs, and so a large pool of specific molecules in cytoplasm are recruited into exosomes. So, the content of exosomal cargo depends on the host cells, and on their cytoplasmatic composition in particular. For adipose-secreted exosome, these specific cargoes are mainly molecules related to the metabolites secreted by adipocyte and lipid storage.
The characterization of exosomes may be carried out by various methods.
1. Nanoparticle tracking analyses can provide the size, size distribution, concentration, and phenotype of the exosome.
2. Dynamic light scattering can provide the diameter range of exosome.
3. Resistive pulse sensing is an essential method which is most useful for investigating cellular function and uptake, and it works when measuring the size distribution and concentration of exosome.
4. Transmission electron microscopy is widely used for the visualization of exosome and is used to study the structure, morphology and size of exosome biological components.
5. Flow cytometry is used to study exosomal surface proteins.
6. Raman spectroscopy can differentiate exosome as a function of the membrane lipid/protein content along with other various surface modifications.
Best.
Mass spectrometry (MS) is indeed a powerful technique for proteomic analysis, including the study of exosomal proteins
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