Updated FTP location. The small GTPase Rab5 has an essential role in sorting vesicles arriving to and leaving the early endosome. One key function of Rab5 in this process is the activation of the primordial phosphatidylinositol 3-kinase VPS34, a lipid kinase that phosphorylates phosphatidylinositol (PI) to generate PI3P. Human VPS34 forms two heterotetrameric core complexes known as complexes I and II. Rab5a preferentially activates endocytic complex II in a membrane dependent manner. To map Rab5a-GTP interaction sites in complex II, unnatural amino acid mediated crosslinking was employed by genetic code expansion. A Rab5a mutant, in which the S84 in was replaced by BrCO6K was used for crosslinking mass spectrometry analysis. BrCO6K is a lysine derivative that can crosslink to several amino acids such as cysteines, histidines, lysines and glutamates over distances up to 15 Å (Cigler et al., 2017; Nguyen et al., 2018). Both complex II and VPS34 alone incubated with Rab5a-GTP-BrCO6K gave rise to a cross-linked product that ran slightly above the 120 kDa molecular weight marker, consistent with 102 kDa VPS34 crosslinked to the 24 kDa Rab5a (126 kDa). The gel bands of this crosslinking product in both samples were cut and analysed by LC-MS/MS for identification of crosslinks.