The study was conducted to elucidate potential cleavage sites of GPR125 which are known to undergo N-terminal shedding through autoproteolysis in a region different to other GRPs. To do these different enzymatic strategies as well as enrichment of both N-terminal and C-terminal peptides, was conducted on media from GPR125 overexpressing HEK cells. Other sample types such as media from non-transfected HEK cells and non-PNGnase treated media showed barely any signal from GPR125. GluC was also used as the common clevage site is located in a region devoid of R/K residues. However, few GPR125 originating peptides were identified from these samples compared to Trypsin and TrypN treated samples. Lysate was also briefly analysed resulting in peptides spanning more of the C-terminal end of the protein which were not previously found in the up-concentrated media.