Cells produce an unexpected high number of protein variants that start at sites that differ from the anticipated start site. Most of these amino(N)-terminal variants or proteoforms are shorter than the expected protein, though some carry N-terminal extensions when ribosomes start translating from sites present in the annotated 5’ untranslated regions of transcripts. Close to one-fifth of all identified protein N-termini in higher eukaryotes point to the existence of such N-terminal proteoforms, thus leading to even more complex proteomes than anticipated, and a major question emerging now is on the functional impact of these N-terminal proteoforms. We previously demonstrated that N-terminal protein variants made from the same gene may have different stabilities in cells. We now hypothesize that N-terminal proteoforms might end up in different, yet unexplored protein complexes. To assess this, we will start by constructing a comprehensive catalogue of N-terminal proteoforms expressed by a human cell line. Secondly, bioinformatics will be used to select up to 100 human genes that encode different N-terminal proteoforms. Third, a novel technology for assessing protein-protein interactions, Virotrap, will be used and further developed for characterizing the interactomes of these proteoforms. Finally, selected interactions will be validated in appropriate models and the functions of N-terminal proteoforms will be investigated at the cellular level.