Methods for proteomics-based analysis of the human muscle secretome using an in vitro exercise model

Over the last decade, the skeletal muscle as a secretory organ gained in importance. A growing number of peptides are described which are produced and released by the muscle fibers and work in an autocrine, paracrine, and endocrine fashion. The contraction-induced secretion of these myokines is considered to contribute to the health-promoting effects of exercise. To gain further insights into the molecular processes that occur during contraction an in vitro exercise model, electric pulse stimulation (EPS), was established. Recent publications show that this model is suitable to electro-stimulate human skeletal muscle cells and thus mimic muscle contraction in vitro. Here, we provide a detailed protocol for the proteomics-based analysis of the human muscle secretome, starting with the cultivation of human myotubes and their electric pulse stimulation, ending with sample preparation for targeted and untargeted proteome analysis of the cell culture supernatant. This whole workflow should allow deeper insights into the complex nature of the muscle secretome and the identification of new myokines which might help to understand the crosstalk of the working muscle with different organs and the beneficial effects of exercise.