Enhanced secretomics workflow reveals organ-dependent secreted protein landscape across multiple murine tissues

Exercise exerts benefits across multiple organ systems and is associated with prevention and treatment of many diseases, including cardiovascular disease, pre-eclampsia, and type 2 diabetes mellitus, among others. Despite this, the identity, origin, and mechanism(s) of action of molecules facilitating these beneficial outcomes are relatively unexplored. The interrogation of the exercise- and organ-dependent secretome is essential to understanding the origin, destination, and therapeutic potential of exerkine-mediated inter-organ signaling. However, comprehensive organ-dependent secretomics faces several analytical challenges, including large dynamic range, blood contamination, and risk of cell burst during preparation. Further, unconventional protein secretion pathways make bioinformatic prediction of all secreted proteins challenging, resulting in poor proteome annotation and requiring robust validation of secreted candidates.

We have developed a robust workflow for the interrogation of organ-dependent secretomes from murine tissue. Tissues are dissected within minutes of animal sacrifice and are rapidly rinsed with PBS buffer to remove residual blood. We implement a low-speed filtered centrifugation to remove the extracellular fluid. Fluid is centrifuged to remove cells followed by albumin/IGG depletion, and then further separated via ultra-centrifugation or magnetic beads to distinguish between extracellular vesicles and released proteins. All samples are analyzed with LC-MS/MS and quantified via TMT.

We applied this protocol to iWAT, skeletal muscle, lung, and placenta. Both the lung and placenta produce the highest volume of EF of 10 tested murine tissues; both fluids had protein concentrations of >50 ug/uL, producing >700 ug of protein per sample, on average. While the lung is amenable to our secretomics method without modification, the placenta required a slightly modified procedure with extra perfusion. Similarly, the iWAT tissue required a brief fixation in paraformaldehyde. Western blots show that these fluids are distinct from organ lysates and lack markers that would indicate cell breakage and/or other contaminant infiltration.

LC-MS/MS analysis revealed >3000 proteins in the extracellular fluid as well as the extracellular vesicles, with unique proteins present in each fraction that reinforce the need for extracellular fluid fractionation. Comparing sedentary and exercised mice revealed lung specific cytokines present only in the lung EF, including the CXL15, an inflammation-induced neutrophil trafficking protein. Additionally, MCK-PGC1alpha transgenic mice show distinctions in secreted proteomes among their muscle, lungs, and placenta, emphasizing the advantage of our methods for the characterization of physiology-dependent signaling. Overall, this optimized secretome isolation procedure has the ability to reveal dynamic changes in organ-dependent signaling that can further enhance therapeutic discovery efforts.