TRAINSPOTTER: profiling nascent protein N-termini indicative of bacterial translation initiation via deformylation-assisted N-terminomics.

Accurate delineation of bacterial translation initiation sites (TISs) remains a major challenge, as conventional genome annotation and ribosome profiling (Ribo-seq) often lack the resolution to discriminate closely spaced start codons. To overcome these limitations, we developed TRAINSPOTTER (TRAnslation INitiation SPOTTER), a deformylation-assisted N-terminomics workflow that enables direct, proteome-wide detection of nascent N-termini indicative of active translation initiation. TRAINSPOTTER exploits the universal N-terminal formylation of initiator methionine in bacteria: in vitro enzymatic deformylation by peptide deformylase (PDF) generates a diagnostic hydrophilic shift, allowing selective isolation of previously formylated, initiation-derived peptides by COFRADIC-based chromatography. Optional in vivo PDF inhibition transiently enriches formylated N-termini, primarily enhancing detection sensitivity. Integration of pulse SILAC (Stable Isotope Labeling by Amino acids in Cell Culture) labeling confirmed that deformylation-shifted peptides represent newly synthesized N-termini, validating TRAINSPOTTER's specificity for nascent translation products. Application to Escherichia coli enabled precise mapping of >1000 TIS-indicative N-termini, including numerous alternative and near-cognate start sites, providing direct proteomic evidence for co-expressed N-terminal proteoforms. The method complements and refines Ribo-seq datasets, offering amino acid-level resolution for otherwise ambiguous initiation events. TRAINSPOTTER thus establishes a robust biochemical framework for proteome-wide identification of TISs and advances the experimental annotation of bacterial proteomes.