biostudies-literatureGarcia-Del Rio DFUniversity of LilleFonds Wetenschappelijk OnderzoekInstitut National de la Santé et de la Recherche MédicaleUniversité de Lille105943https://www.ebi.ac.uk/biostudies/studies/S-EPMC9971881biostudies-literatureUnknown26(2)Eukaryotic mRNA has long been considered monocistronic, but nowadays, alternative proteins (AltProts) challenge this tenet. The alternative or ghost proteome has largely been neglected and the involvement of AltProts in biological processes. Here, we used subcellular fractionation to increase the information about AltProts and facilitate the detection of protein-protein interactions by the identification of crosslinked peptides. In total, 112 unique AltProts were identified, and we were able to identify 220 crosslinks without peptide enrichment. Among these, 16 crosslinks between AltProts and Referenced Proteins (RefProts) were identified. We further focused on specific examples such as the interaction between IP_2292176 (AltFAM227B) and HLA-B, in which this protein could be a potential neiScienceEmploying non-targeted interactomics approach and subcellular fractionation to increase our understanding of the ghost proteome.PMC9971881G008018NBonnefond AGevaert KGarcia-Del Rio DFSalzet MFournier ICardon TEyckerman SfalseEmploying non-targeted interactomics approach and subcellular fractionation to increase our understanding of the ghost proteome.Eukaryotic mRNA has long been considered monocistronic, but nowadays, alternative proteins (AltProts) challenge this tenet. The alternative or ghost proteome has largely been neglected and the involvement of AltProts in biological processes. Here, we used subcellular fractionation to increase the information about AltProts and facilitate the detection of protein-protein interactions by the identification of crosslinked peptides. In total, 112 unique AltProts were identified, and we were able to identify 220 crosslinks without peptide enrichment. Among these, 16 crosslinks between AltProts and Referenced Proteins (RefProts) were identified. We further focused on specific examples such as the interaction between IP_2292176 (AltFAM227B) and HLA-B, in which this protein could be a potential ne2023-01-01T00:00:00Z2023 Feb2026-05-28T08:10:49.557Z2025-02-19T02:21:42.642ZS-EPMC99718813686604110.1016/j.isci.2023.105943