Exosomes are 30-150 nm extracellular vesicles that are endocytically released from cells. Exosomes were previously thought to simply exude unnecessary proteins from cells; however, more recent research has highlighted their importance in cell-to-cell communication by transporting biomolecules such as mRNA, microRNA, and protein through the circulatory system. Recipient cells may then take up these molecules, resulting in modulation of a wide variety of cellular functions. Notably, exosomes have been shown to play a role in antigen presentation and transportation of infectious materials from one cell to another.
Exosomes are secreted by most cell types and are found abundantly in many body fluids. As such, their potential as a source of diagnostic biomarkers is an area of intense interest. In fact, efforts are already underway to catalog exosomal proteomes. Advancements in proteomic tools, along with improvements in exosomal purification techniques, have contributed immensely to this effort. However, despite the past decade’s explosion of interest in exosome biology, our understanding of its role in normal cellular physiology is currently sparse.
Some exploratory studies aiming to profile exosomal protein biomarkers have exploited high-content techniques, including antibody arrays and mass spectrometry. For example, Jaworski et al. employed the RayBio Cytokine Array 1 to compare the cytokine profiles of exosomes released from T-cell lines that were infected with the human T-lymphotrophic virus type 1 (HTLV-1) with those from T-cells that were uninfected. They observed that exosomes from different cell lines had distinct cytokine profiles, with exosomes from HTLV-1 infected cells having elevated levels of GM-CSF and IL-6 and reduced levels of MCP-1 and RANTES. Their data suggested that exosomes may play a role in cytokine signaling during HTLV-1 infection. In a study by Lai et al., the proteomic profile of exosomes released from huES9.E1 cells, a human ESC-derived mesenchymal stem cell line, was analyzed using a RayBio Label-based Array targeting 507 human proteins.
Interestingly, both studies employed mass spectrometry to characterize the protein profile of exosomes in addition to antibody array analysis. In Jaworski’s study, LC-MS/MS analysis was unable to detect any cytokine with high confidence. In Lai’s experiment, the antibody array detected 91 proteins that were not detected by mass spectrometry. Their studies highlight the advantages of antibody arrays, which include:
- No risk of ion suppression. Ion suppression is a phenomenon linked to electrospray ionization mass spectrometry, which negatively affects the detection of certain species. No such phenomenon occurs with antibody arrays.
- Independent of protein concentration. Without depleting samples of high abundance proteins, mass spectrometry analysis may not detect medium and low abundance proteins. However, the detection of medium and low abundance target proteins by antibody arrays is not affected by the presence of high abundance proteins.
- Less sample manipulation. Bottom-up mass spectrometry, the most common type of mass spectrometry method, requires that the proteins are denatured, reduced, and digested prior to analysis. This procedure often occurs over a day. On the other hand, proteins that are extracted from exosomes can be directly added to antibody arrays.
- Affordable equipment that requires minimal training. Mass spectrometers are expensive, and operators of mass spectrometers must have specialized and in-depth training. Antibody arrays, if using a membrane solid support, can be analyzed using a Western blot imager, a common laboratory instrument, for which no specialized training is required. Minimal training is also needed to operate laser scanners for analyzing glass-based antibody arrays. For those without access to a compatible laser scanner, RayBiotech will scan and extract the data of RayBiotech glass arrays at no extra charge.
- Flexible. Like mass spectrometry, antibody arrays can be used to analyze the protein levels within a specific subset of proteins (e.g., cytokines) or across a wide range of protein types.
Jaworski, Elizabeth, et al. “Human T-lymphotropic virus type 1 infected cells secrete exosomes that contain tax protein.” Journal of Biological Chemistry (2014): jbc-M114
Lai, Ruenn Chai, et al. “Proteolytic potential of the MSC exosome proteome: implications for an exosome-mediated delivery of therapeutic proteasome.” International journal of proteomics 2012 (2012).