Jennifer Geddes-McAlister

As the American Chemical Society marks its 150th anniversary, it is a fitting moment to reflect on how a young analytical discipline, proteomics, matured into a central pillar of the chemical and biological sciences and how the Journal of Proteome Research (JPR) chronicled and guided that transformation. Founded at the dawn of shotgun proteomics, JPR has published pivotal advances in separation, ionization, computational interpretation, quantitative strategies, and biological applications. In this Perspective, we trace that arc from the first near-complete eukaryotic proteomes through the quantitative and post-translational era, the computational turn, and the current high-throughput, integrative, and artificial-intelligence-assisted phase. Along the way, we highlight how the same infrastructure enabled mass spectrometry-based metabolomics, yielding an integrated framework for molecular phenotyping.

Protein-protein interactions (PPIs) influence critical biological processes in pathogenic microorganisms, such as the human fungal pathogen, Cryptococcus neoformans . Fungal thermotolerance and stress response pathways are key virulence determinants that directly impact pathogen adaptation and survival and the infection process. To establish a comprehensive baseline of PPIs in C. neoformans and explore these interactions to infer functional roles for uncharacterized proteins, we applied size exclusion chromatography coupled with mass spectrometry to the secreted and cellular proteomes of the fungi. As a result, 216 and 1699 unique proteins were identified across 24 secretome and proteome fractions, respectively. The predicted secretome networks included expected proteins associated with vesicles and virulence, indicating a role in extracellular defense. Whereas the cryptococcal proteome highlighted interactions among proteins with defined roles in fungal virulence for protein stability and thermotolerance, including two previously uncharacterized proteins, CNAG_00287 and CNAG_05199, putatively involved in complex formation with heat-shock proteins (HSP). Based on sequence and structure homology, we propose that CNAG_00287 is a tetratricopeptide repeat-containing co-chaperone that modulates Hsp 70 activity and CNAG_05199 functions as a Hsp70. We validated the thermotolerance role of CNAG_00287 in heat-related stress, as its absence significantly impaired fungal growth in nutrient-limited media at 37 °C. Together, this work resolves virulence-associated PPIs within C. neoformans and reveals new molecular regulators of thermotolerance that underpin fungal pathogenicity.

Efficient and reproducible protein extraction is a critical step in mass spectrometry-based proteomics workflows, particularly for complex host–pathogen systems where low-abundance immune-associated proteins are difficult to detect. Probe sonication methods used for cell lysis requiring mitigation of excessive heat generation, to prevent degradation of biologically important proteins, while also limiting throughput and potentially introducing sample-to-sample variability. In this study, we evaluated adaptive focused acoustics (AFA) technology as an alternative approach for macrophage lysis and protein extraction and digestion within a standard proteomics workflow coupled with mass spectrometry. We observed that AFA technology reduced hands-on processing times and overall workflow timelines and single-sample AFA technology improves proteome coverage, dynamic range, and reproducibility. We also evaluated multiplexed AFA technology for lysis, and we observed an exclusive macrophage proteome and influence on replicate reproducibility and dynamic range detection for low abundant proteins. Moreover, multiplexed AFA technology for macrophage lysis and digestion further increased protein identifications, replicate reproducibility, and dynamic range. Considering the AFA-exclusive proteome, 86 proteins were detected across all AFA-based lysis and digestion methods, including low-abundance proteins associated with macrophage homeostasis, inflammatory response, and transport. Together, these findings demonstrate that AFA technology enhances reproducibility, throughput, and proteome depth for macrophage protein extraction while enabling the detection of biologically relevant low-abundance immune-associated proteins. These improvements provide a strong foundation for future investigation of host–pathogen infection models, where pathogen-derived proteins remain challenging to detect within complex host proteomes.

Iron is an essential nutrient that underpins fundamental biological processes, yet its bioavailability is severely restricted during infection due to oxidation and host-mediated sequestration. In Gram-negative pathogens, such as Klebsiella pneumoniae, iron limitation imposes a critical selective pressure, necessitating tightly regulated acquisition systems to support growth, virulence, and survival. While canonical pathways for iron uptake are well characterized, regulatory mechanisms coordinating these processes remain incompletely understood. Here, we applied mass spectrometry-based phosphoproteomics to identify iron-responsive regulatory events associated with bacterial iron homeostasis. This approach revealed iron-dependent phosphorylation of SbmA, a conserved inner membrane transporter previously implicated in the uptake of antimicrobial peptides and related substrates. Functional characterization demonstrated that deletion of sbmA results in reduced intracellular iron levels and altered cellular morphology, supporting a role in iron acquisition. Complementary proteome mapping of δsbmA revealed compensatory production of siderophore receptors and TonB-dependent transport systems, further implicating SbmA in maintaining iron balance. Leveraging these findings, integration with high-throughput drug screening identified a compound that exploits SbmA-mediated transport to inhibit bacterial growth, highlighting its potential as a therapeutic entry point. Collectively, this work uncovers a previously unrecognized role for SbmA in iron homeostasis and demonstrates the power of phosphoproteomics to identify condition-specific regulators of essential bacterial pathways. These findings position SbmA as a promising target for antimicrobial development in K. pneumoniae.

Klebsiella pneumoniae is an opportunistic bacterial pathogen associated with high morbidity and mortality, exacerbated by the rapid emergence of resistance to last-resort antibiotics, such as carbapenems. Adaptation to nutrient limitation, particularly fluctuations in metal availability, is critical for bacterial survival and virulence, yet the regulatory mechanisms coordinating these responses remain incompletely understood. Protein phosphorylation represents a key post-translational modification governing bacterial physiology and offers a promising avenue for identifying novel antimicrobial targets. Here, we applied mass spectrometry-based phosphoproteomics to define nutrient-responsive signaling networks in K. pneumoniae under varying iron and zinc conditions. This analysis identified iron-dependent phosphorylation of TolQ, a conserved inner membrane component of the Tol-Pal system that maintains cell envelope integrity. Structural modeling predicted that phosphorylation modulates TolQ-TolR conformation, suggesting a mechanism by which iron availability regulates Tol-Pal function. Functional characterization demonstrated that deletion of tolQ results in reduced bacterial viability, increased susceptibility to host immune clearance, and heightened sensitivity to antibiotic treatment. To further explore the therapeutic potential of this pathway, we integrated high-throughput compound screening with computational modeling and identified small molecules that phenocopy δ tolQ . Collectively, these findings reveal a previously unrecognized link between iron availability and phosphoregulation of the Tol-Pal system and establish TolQ as a critical mediator of bacterial survival. This work highlights phosphoproteomics as a powerful strategy to uncover regulatory vulnerabilities and identify targets for antimicrobial development in drug-resistant pathogens.

Summary: Fusarium head blight (FHB) is a major threat to global wheat production and food safety due to contamination with mycotoxins, such as deoxynivalenol (DON). The emergence of new mycotoxin chemotypes, including 7-α-hydroxy,15-deacetylcalonectrin (3ANX), presents an evolving challenge for disease management and resistance breeding. Here, we performed a field-based, systems-level proteome analysis of wheat infected with Fusarium graminearum strains belonging to the common 15ADON and recently emerged 15ADON/3ANX chemotypes. Across host and pathogen, we quantified more than 9,200 proteins, providing extensive coverage of infection-associated molecular responses. Infection with 15ADON/3ANX strains suppressed canonical wheat detoxification pathways while promoting structural and oxidative defence responses. Concurrently, the fungal proteome of 15ADON/3ANX-producing strains indicated altered mitochondrial ribosome function and alternative virulence strategies. Further investigation of the host-pathogen interface defined hub protein networks negatively regulating classical detoxification markers, suggesting coordinated regulation of host defence responses regardless of chemotype. Molecular responses were linked to field phenotypes by quantification of DON-3-glucoside/DON ratios and disease severity, defining positive correlations in 15ADON infections, which were abolished upon 15ADON/3ANX infection, indicating chemotype-specific evasion or suppression of host defenses. These findings demonstrate reprogramming of host–pathogen interaction networks and reveal molecular targets that may inform chemotype-aware breeding strategies to enhance crop resilience.

Fungal infections are a major global health challenge, with current antifungal therapies limited by toxicity, cost, and resistance. For Cryptococcus neoformans , key virulence factors that initiate and sustain infection are regulated by fungal peptidases to produce a polysaccharide capsule, promote immune evasion, and support antifungal resistance. These peptidases represent promising targets for antivirulent therapeutic strategies. Here, we developed a computational pipeline to predict and design peptide- and protein-based inhibitors against cryptococcal peptidases. Specifically, we targeted three virulence-associated peptidases: Rim13 (cysteine), May1 (aspartic), and CnMpr1 (metallo). Cysteine peptidase inhibition decreased capsule/cell size ratios without impeding fungal growth and reduced fungal survival within macrophages. Similarly, aspartic peptidase inhibition enhanced fungal clearance within alveolar macrophages and disrupted biofilm formation with additive effects towards fluconazole susceptibility in resistant strains. Additionally, metallopeptidase inhibition through catalytic zinc chelation and blocked substrate binding led to enhanced enzymatic inhibition and reduced in vitro blood-brain barrier crossing. Moreover, an in vivo larval model assessing inhibitor efficacy produced additive effects with fluconazole and lacked host cell cytotoxicity and fungicidal properties, reinforcing anti-virulence mechanisms and therapeutic potential while limiting the evolution of resistance. Further, global proteome profiling of inhibitor treated cells defined a mechanism of cell wall disruption, impeding fungal virulence. Taken together, the designed peptidase inhibitors exhibited potent antifungal activity without harming mammalian cells, establishing a predictive framework for rational scaffold design of next-generation antifungals that disarm the pathogen enabling immune-mediated clearance.

Lon protease is a global regulator in bacteria affecting biofilm formation, antibiotic resistance, toxin-antitoxin complexes, and proteolysis of damaged proteins. In Klebsiella pneumoniae , Lon has roles in iron response, protein transport, and membrane assembly. Here, we apply bottom-up proteomics to identify proteins in the extracellular environment of WT and Δ lon K. pneumoniae .