Intact extracellular vesicle cross-linking on vesicles derived from MDA-MB-231 and LM2 cells

The development of any pharmaceutical requires characterization of the compound, usually be several techniques. Therefore, characterizing the protein content and biochemical state of EVs is of great importance for their useif they are to be used as biopharmaceuticals. Structural characterization of proteins in EVs remains a great challenge even for state-of-the-art technologies such as super-resolution and electron microscopy, partly due to the biophysical and biochemical heterogeneity of EVs. Here we overcome some of these challenges by using cross-linking mass spectrometry (XL-MS) to structurally characterize protein complexes and the interactome of intact EVs, at an unprecedented resolution of up to 30Å. By applying XL-MS to intact EVs derived from two metastatic breast cancer cell lines (MDA-MB-231 and LM2), we validate the presence of active forms of protein complexes that are known to play a role metastasis, revealing for example previously uncharacterized regions of Moesin. Moreover, a cross-linking data-driven molecular docking supports novel conformations of ANXA2 in LM2 EVs.