Hunter-Dia: An updated protocol for enrichment and mass spectrometry-based identification of protein N-terminal...

Protein N-termini encode essential biological information, reflecting not only the identity of the translation start site but also a range of co- and post-translational modifications (PTMs), including N-terminal acetylation, myristoylation, and proteolytic processing events. These modifications are critical for regulating protein stability, localization, and function. However, standard bottom-up proteomics workflows typically focus on internal tryptic peptides and often fail to comprehensively capture N-terminal peptides, leading to an underrepresentation of the N-terminome in global proteomic datasets. To address this gap, N-terminal peptides can be enriched by negative selection, which modifies free primary amines before proteome digestion to enable simultaneous enrichment endogenously modified and protease-generated N-terminal peptides by depletion of internal and C-terminal peptides. Here we present an updated, 2-day step-by-step protocol for High-efficiency Undecanal-based Enrichment of N-termini (HUNTER) combined with Data-Independent Acquisition (DIA) mass spectrometry for deep and reproducible N-terminome profiling. To support broad adoption, we provide pre-configured FragPipe and DIA-NN search templates optimized for N-terminomics data, as well as an open-source R/Quarto pipeline for automated downstream analysis. This includes annotation of cleavage sites and PTMs, classification of native and protease-generated neo-N-termini, and statistical analysis of differential abundance across conditions. HUNTER-DIA consistently achieves high N-terminal labeling and internal peptide depletion efficiencies, enabling sensitive detection of endogenous processing events and dynamic N-terminal PTMs across diverse sample types. This platform opens new opportunities for studying protease biology, N-terminal post-translational modifications, and their context-specific regulation in health and disease across virtually any organism.