Molecular profiling of tumor-derived exosomes in plasma of patients with melanoma

By isolation of exosomes from body fluids such as serum or plasma with the use of a gel filtration method developed for this purpose, we showed that the level of exosomes in patients with melanoma is higher and may be associated with progression of the tumor and treatment response. By applying the techniques of mass spectrometry we would like to assess the protein profile of exosomes released by melanoma cells and determine whether these changes correlate with selected clinical parameters. The model tests are exosomes released in vivo from melanoma cells (MMTEX). Proteins of exosomes were subjected to qualitative and quantitative analysis by LC-MS/MS. Proteins were digested by trypsin and were subsequently purified on C18 StageTips which significantly improves the chromatographic separation and allows identification of a higher number of proteins. In addition, after digestion the obtained peptides were fractionated and separated using ion chromatography and reversed-phase chromatography (C18 column), and then analysed by tandem mass spectrometry analysis. To evaluate protein content and verify the presence of exosomes we were used the Western blot technique or flow cytometry.