Interrogation of bacterial pathogen canonical and non-canonical phosphorylation events - Abstract Over the past three decades, significant progress in canonical phosphoproteomic analysis has been driven by immobilized metal affinity chromatography (IMAC), which enables enrichment of serine-, threonine-, and tyrosine-phosphorylated peptides following enzymatic digestion of cellular samples. While effective, IMAC methods rely on harsh buffer conditions and often suffer from limited specificity and reproducibility. In contrast, non-canonical phosphorylation occurring on residues such as histidine, aspartate, glutamate, cysteine, lysine, and arginine remain poorly understood due to the lack of robust enrichment strategies. These underexplored post-translational modifications (PTMs) have been increasingly implicated in critical cellular processes, including signal transduction, energy metabolism, and adaptive responses. Despite their biological importance, both canonical and non-canonical phosphorylation events lack efficient, selective tools for enrichment to allow comprehensive detection, representing a major bottleneck to advance the field. To address these limitations, we have recently developed a new class of affinity reagents, phosphopeptide “SuperBinders”, that are based on engineered SH2 domains and are capable of selectively enriching canonical phosphotyrosine peptides under mild pH conditions. These reagents overcome key limitations of traditional IMAC methods, offering improved recovery, specificity, and compatibility with downstream mass spectrometry workflows. In parallel, we have also developed a high-affinity phosphohistidine-specific SuperBinder, derived from a short-chain Fab fragment, to enable the enrichment and detection of histidine-phosphorylated peptides, representing a novel and technically innovative approach to interrogating non-canonical phosphorylation. This application seeks to advance these SuperBinders as next-generation tools for comprehensive phosphoproteomic analysis. Specifically, we will optimize the technical application of these protein-based reagents, benchmark their performance, expand their application to diverse biological systems, and ultimately develop protocols for their broad adoption. As proof of principle, we will apply these tools to mammalian and bacterial cells to investigate phospho-signaling networks. Using Escherichia coli (E. coli) and Mycobacterium tuberculosis (Mtb), we will define the dynamic remodeling of canonical and non-canonical PTMs in response to environmental stress and targeted pharmacological perturbation, respectively. In Mtb, we will further interrogate phosphorylation events associated with response to novel antibacterial compounds to elucidate mechanisms of action. Our approach represents substantial technical innovations in phosphoproteomics, with broad implications for advancing PTM biology, uncovering new regulatory pathways, and accelerating therapeutic discovery.
