Proteomic profiling of extracellular fluids to identify secreted proteins from muscle and fat tissues

Communication between tissues or different cells within a tissue is often a result of secreted molecules such as metabolites, lipids, nucleic acids, or proteins (referred to as the secretome). These enter the extracellular space and may subsequently pass into the circulation. Depending on their nature, concentration and context, these molecules initiate specific responses in their target cells. Environmental stimuli such as exercise and cold exposure, but also different diseases, are known to significantly alter the secretome and thereby affect whole body homeostasis. Thus, identifying these factors is of great interest. The analysis of secreted proteins, however, represents a unique challenge for the field. This is mainly because mass spectrometry can be limited by the dynamic range problem, whereby the detection of low abundance polypeptides can be masked by the presence of high abundance proteins. Plasma, muscle, and fat all contain specific proteins of very high abundance, making it tremendously challenging to detect low abundance proteins in these biological samples. Thus, secreted, hormone-like polypeptides frequently remain undetected. Because muscle and fat are known to communicate by secretion of myokines and adipokines, respectively, we have sought to develop methods that can circumvent these issues through the isolation of extracellular fluids (EF) which surround these tissues. EFs had previously been isolated for analysis of metabolites; however, whether this method could be made useful for in depth proteomics analysis was not known. Recently, we have developed a method that modifies these procedures and makes it applicable for the study of EF proteins. We have applied this to muscle and fat EFs, but in principle, it can be used to study secreted proteins from almost any tissue in any species, including humans. A step-by-step protocol and methods of quality control are given below.