Protein-protein interactions (PPIs) regulate every aspect of cell life, playing a pivotal role in terms of accurate governement of multicellular organisms through fine regulation mechanisms. The consistent organization of PPIs networks increases exponentially the cell capability to control a plethora of molecular phenomena, including signal transduction, endocytosis, secretion and even cell functioning or death. However, when this knotty balance between assembly and disassembly is hampered, the effects can impact on organs and tissue functionality.

It is a matter of fact that the identification of PPIs networks as potential pharmacological target in the treatment of several diseases represents a consolidate strategy in personalized medicine. Furthermore, advancements in biochemical field continuously unravel the PPIs complexities, providing deeper insights into cellular function and discovering potential therapeutic candidates.

To dissect that molecular intricacy, cross-linking mass spectrometry (XL-MS) emerged as one of the most promising strategies to capture the dynamics of interactomes from small scale, such as macromolecular complexes to large scale, including vesicles, organelles and cells.

Since the plasma membrane proteins (PMPs) constitute ~60% of all drug targets, have access to the complete "membranome" PPIs network would represent an unbiased approach to depict regulatory pathways controlling cell behavior. Despite the biochemical impact, knowledge gaps regarding protein interactome at PM persist.

In this challenging context, we applied an XL-MS approach combined with PM purification pipeline to profile, on a system-wide scale, PM interactome of He-La cells used as cellular reference model. Specifically, we enriched PM-fractions from total cell lysates cross-linked before (native) and after (disrupted) PM dissolution. We defined the best experimental settings in terms of MS-cleavable cross-linker concentration, cross-linking reaction time, PM enrichment, MS acquisition and elaboration.

Based on all detected inter-links, we assembled a protein global network composed of unique residue-to-residue pairs originating from more than 300 proteins assigned to the PM compartment. Then, we compared our experimental PMPs map to already annotated interactions and we revealed an overlap of 25%, confirming the accuracy of our dataset. With respect to PM-associated proteins, we targeted the highest number of PPIs for specific PM member families, counting ATP-binding cassette transporters, solute carrier transporters family, ATPase Na+/K+, Cu2+ and phospholipid transporters. Finally, we identified the remaining cross-links as part of proteins which are currently not described as interacting with each other, presenting potentially de novo partners.

Currently, we are implementing our XL-MS cell membranome pipeline to unravel the existence of hidden disease-specific protein subnetworks in cell models of genetic metabolic disorders.