Profiling Optimal Conditions for Capturing EDEM Proteins Complexes in Melanoma Using Mass Spectrometry.

Abstract

Endoplasmic reticulum (ER) resident and secretory proteins that fail to reach their native conformation are selected for degradation through the ER-Associated Degradation (ERAD) pathway. The ER degradation-enhancing alpha-mannosidase-like proteins (EDEMs) were shown to be involved in this pathway but their precise role is still under investigation. Mass spectrometry analysis has contributed significantly to the characterization of protein complexes in the last years. The recent advancements in instrumentation, especially within resolution and speed can provide unique insights concerning the molecular architecture of protein-protein interactions in systems biology. Previous reports have suggested that several protein complexes in ERAD are sensitive to the extraction conditions. Indeed, whilst EDEM proteins can be recovered in most detergents, some of their partners are not solubilized, which further emphasizes the importance of the experimental setup. Here, we define such dynamic interactions of EDEM proteins by employing offline protein fractionation, nanoLC-MS/MS and describe how mass spectrometry can contribute to the characterization of such complexes, particularly within a disease context like melanoma.