Unraveling N-Terminal Proteoform Interactomes via Multiplexed Recombineering in Salmonella.

N-terminal proteoforms (Nt-proteoforms), which arise through alternative translation initiation, exhibit variation at their N-termini that can influence subcellular localization, protein stability, and functional roles within multiprotein complexes. Advances in riboproteogenomics, particularly in ribosome profiling, have highlighted the significance of the alternative proteome in bacteria. However, Nt-proteoforms remain largely underexplored, emphasizing the need for detailed protein-protein interaction (PPI) studies to elucidate their potentially differential biological roles.Mass spectrometry-based proximity labeling techniques offer a high-throughput method for PPI screening. Here, we present a multiplexed recombineering approach that enables the genomic integration of a promiscuous biotin ligase (PBL) via double-stranded DNA (dsDNA) recombineering while simultaneously allowing the targeted mutation of specific translation initiation sites (TIS) using single-stranded DNA (ssDNA) oligos through oligo-mediated allelic replacement (OMAR). This multiplexed recombineering approach facilitates endogenous BioID tagging and the identification of Nt-proteoform-specific proxeomes.