Advances in Decoding Bacterial N-Terminal Proteoforms: Technologies, Challenges, and Functional Insights

The bacterial proteome is a highly dynamic landscape rather than a static reflection of the genome. Recent research revealed that proteome complexity extends far beyond canonical gene annotation, with N-terminal (Nt-)proteoforms emerging as an important underexplored additional regulatory layer. These molecular variants originate from a single genetic locus through alternative translation initiation at internal or external in-frame start sites, thereby generating N-terminal heterogeneity that can influence protein stability, subcellular localization, interaction networks, and the stoichiometric assembly of multiprotein complexes. While recent advances in riboproteogenomics, N-terminomics, and computational annotation strategies have enabled proteoform mapping at single-amino acid resolution, rapid high-throughput discovery currently outpaces downstream functional characterization. This review discusses the technological advances driving Nt-proteoform discovery, including emerging ribosome profiling and proteogenomic approaches, and further evaluates strategies for the functional characterization of Nt-proteoform. Particular emphasis is placed on the transition from conventional plasmid-based heterologous expression systems toward precise genome-engineering approaches that enable selective manipulation of alternative translation initiation events within their native genomic context. Such targeted strategies are essential to bridge the gap between Nt-proteoform identification and functional understanding, ultimately uncovering how individual bacterial genomic loci can encode proteoforms with distinct and potentially polarized roles in bacterial physiology and pathogenesis.