In the quest for circulating biomarkers of disease or drug response, much effort has been made to avoid the overwhelming effect of high- abundance proteins in the blood that obscure lower abundance and potentially more interesting proteins. Removal of the high-abundance proteins with multiple affinity antibody columns has been very effective in this regard. An alternative approach that has been successful as well is to isolate plasma proteins as they are being released from the source tissues or cells. One common such method involves harvesting proteins released by tissue culture cells into conditioned media [42,43] . Although this approach has been productive, proteins may be lost due to dilution or adherence to the culture container, and it is generally limited to cultured cells rather than whole tissue. Analysis of tissues can provide a more physiological context, as they include primary cells as well as the natural composition of multiple cell types that may modulate secretion. Tissues can also be taken from the host, typically animal models of disease, following drug treatment, in order to assess the impact of treatment on the secretome. This can lead to a better understanding of the mechanism of action of the drug treatment as well as the identification of possible markers of therapeutic response. Proteins released from tissue into culture medium have also been studied , but contamination from lysed cells and culture medium can obscure the actual proteins secreted. Reliance on those proteins with a signal sequence as well as isotopic labeling of de novo synthesized proteins from the tissue are usually necessary.
An alternative approach to identifying and quantifying secreted proteins relies on the isolation of such proteins directly from the secretory pathway of the cells or tissue. The contents of the Golgi and related secretory vesicles provide a highly concentrated collection of proteins, just prior to their release from the cell [45,46]. This method avoids dilution into the plasma, which would obscure all but the more abundant secreted proteins. Dilution as well as contamination by cell lysis proteins in culture medium is also eliminated as a concern. Tissues can be as easily investigated as cultured cells, with the added advantage that the samples do not need to be viable at the time of analysis. Thus, frozen surgical samples from patients or animal models are amenable to study and comparison, even when samples are collected at different time points. To isolate secretory proteins directly from cell culture or tissue, the homogenized samples are separated on a density gradient to isolate secretory vesicles.
Was this article helpful?
What you need to know about… Project Management Made Easy! Project management consists of more than just a large building project and can encompass small projects as well. No matter what the size of your project, you need to have some sort of project management. How you manage your project has everything to do with its outcome.