Joseph A. Loo

Native mass spectrometry (nMS) is well established for measuring protein masses and stoichiometries using nanoelectrospray ionization (nESI), yet salt adduction and source activation energies can limit routine measurements. In this study, we benchmark submicron quartz nanopipette nESI emitters (<50 nm internal diameter) across four mass spectrometry platforms (quadrupole-time-of-flight, quadrupole-Orbitrap, tribrid-Orbitrap platforms and timsTOF) and a wide protein range (14.5–800 kDa). We analyzed the intrinsically disordered protein alpha synuclein (αS; 14.5 kDa) and holo-myoglobin (17 kDa) with myoglobin further characterized over a range of concentrations (10 μM-1 nM) and capillary voltages to determine concentration ranges and define a gentle operating regime. We additionally observed reduced Na+ adduction and preservation of the Zn2+-bound metalloproteoform of carbonic anhydrase II (29 kDa). Proteins and protein complexes spanning the mid-to-high mass range including ovalbumin (∼44 kDa), malate dehydrogenase (∼70 kDa), transferrin (80 kDa), glutamate dehydrogenase (∼350 kDa), β-galactosidase (∼465 kDa), and GroEL (∼800 kDa) were readily detected using nanopipette emitters. Compared with conventional 1–2 μm internal diameter borosilicate emitters, quartz nanopipettes provided higher signal-to-noise ratios and fewer adducts. Finally, direct analysis of a clarified bacterial lysate expressing αS yielded a clear monomeric charge-state distribution, demonstrating compatibility with complex biological matrices. Collectively, these results establish quartz nanopipette nESI as an instrument-portable, salt-tolerant approach suitable for routine nMS analysis across a broad range of protein molecular weights and sample complexities.

Iron is an essential micronutrient for nearly all forms of life, including pathogenic microbes that must acquire it from their host during infection. At the host–pathogen interface, humans restrict microbial access to iron through nutritional immunity, which many pathogens overcome by secreting hemophores that scavenge extracellular heme (iron protoporphyrin IX). However, identifying hemophores and other ligand-binding proteoforms in complex proteomes remains challenging using conventional peptide-based bottom-up mass spectrometry (MS). Here, we introduce ProteoMIX (Proteome Analysis by Mixing), a function-based native top-down proteomics workflow that combines slow-mixing mode native MS with charge reduction to identify ligand-binding proteoforms directly from complex mixtures. Applying ProteoMIX to the Corynebacterium diphtheriae exoproteome identified ChtA30–314, an abundant soluble hemophore generated by proteolytic processing of the surface-exposed ChtA heme receptor. Using native top-down MS sequencing, cell fractionation, and gene deletion, we show that the protease DIP2069 releases ChtA30–314 by removing ChtA’s transmembrane helix, producing a soluble proteoform that delivers heme to support microbial growth. In contrast, the related paralog ChtC is not processed, enabling C. diphtheriae to generate localization-specific heme-binding proteoforms from related gene products. Extending this approach to Staphylococcus aureus, ProteoMIX also revealed soluble IsdA hemophores that coexist with surface-anchored variants, demonstrating the generality of the method and suggesting that protease-mediated hemophore release may operate across Gram-positive pathogens.

Gram-positive bacteria display virulence-associated pili that facilitate adhesion and biofilm formation. These pili are covalently polymerized by class C sortase enzymes, which selectively recognize their cognate pilin substrates amid numerous cell wall sorting signal (CWSS)-bearing proteins. The molecular basis for this stringent substrate specificity has remained unclear. Here, we develop a rapid, quantitative fluorescence-activated cell sorting assay to monitor pilus assembly in Corynebacterium diphtheriae , enabling high-throughput analysis of SpaA pilin and SrtA sortase variants. Using this platform, together with molecular modeling and dynamics simulations, we show that SrtA engages nearly the entire SpaA CWSS to form a membrane-embedded complex that incorporates not only the LPXTG motif but also its connector and transmembrane helix elements. Formation of this interface displaces an inhibitory active-site lid and activates the enzyme to load the pilin substrate. Systematic CWSS swapping experiments and deep mutational scanning further support this model, demonstrating that noncognate pilins are excluded because they fail to form the required interface. Conversely, SrtA variants with an artificially unlatched lid bypass the need for this interface, indicating that membrane-driven complex formation is important for substrate licensing. Together, these findings define a “two-factor authentication” mechanism for pilus assembly in gram-positive bacteria: class C sortases first verify pilin identity by forming a membrane-embedded interface that activates the enzyme, then they recognize the LPXTG motif to initiate loading and crosslinking. This work provides a unified molecular framework for selective pilin incorporation in gram-positive bacteria and identifies potential vulnerabilities in the licensing machinery that may be exploited therapeutically.

Supplementing standard electrospray ionization (ESI) solvents with specific low-volatility organic compounds (e.g., sulfolane or any positional isomer of nitrobenzyl alcohol) increases biomolecular analyte charge for mass spectrometry in the phenomenon known as supercharging. Controversial mechanisms responsible for increasing charge are considered, and the data is found to correlate highly to solvent leveling; i.e., protonated solvent is the strongest acid in a solution because any stronger acid simply dissociates to protonate more solvent. Hence, the recipe for increasing charge in positive ion mode is to make the protonated solvent into a stronger acid (equivalent to reducing the neutral solvent’s basicity). That change is accomplished by adding involatile, weak bases to the solvent. A secondary effect of weak base additives is to suppress the solution-phase ionization of weak acid residues; e.g., reducing opposite charging. Here the abilities of analogous compounds to increase or decrease charging in positive ion mode ESI are predicted from experimentally measured basicities. Consistently, amides, nitriles, and pyrazoles more basic than water reduced the average charge of protein analytes electrosprayed from denaturing solutions, while analogues less basic than water increased the average charge, establishing the veracity of solvent leveling as a supercharging mechanism. In other words, reducing the charge departing on solvent leaves more charge for the protein analyte.

Herein, we demonstrate how Au(III) organometallic chemistry offers the potential to streamline the generation of constrained peptide tricycles. By leveraging the chemoselectivity and ultrafast kinetics of tetrametallic Au(III)-based aryl reagents toward cysteine (Cys) bioconjugation, we have constructed a small library of constrained peptide tricycles. These tricycles can be synthesized and isolated in 25-55% yields from various tetracysteine-containing linear peptides. In all cases, tricyclization conversion occurs quantitatively under five min at sub-mM concentrations of the target peptide and proceeds under varied distributions of the four Cys residues in the target peptide sequence. Furthermore, by constraining various linear peptides around the aryl core of tetraphenylethylene, a common solid-state luminogen, we generate fluorescent macrocycles with robust solution-state emissive profiles. We evaluated the innate affinity of these fluorescent hybrid peptides toward cells, finding that they behave as highly selective luminogenic cell markers of cellular debris.

Staphylococcus aureus extracts hemin from human hemoglobin (Hb) to overcome host-imposed iron limitation. How it recovers Hb-bound hemin from the hemoglobin:haptoglobin (Hb:Hp) complex, the major circulating form of Hb outside red blood cells, remains unclear. Here we use cryo-electron microscopy, biophysical measurements, and solution kinetics to define how the S. aureus IsdH surface receptor extracts hemin from Hb:Hp. A 3.1 Å cryo-EM structure of Hb:Hp bound by full-length IsdH reveals that its N-terminal NEAT domain (N1) anchors it to αHb, whereas its downstream N2N3 extraction unit engages βHb to remove its hemin. The receptor engages Hb:Hp differently than isolated Hb, because N-linked glycans on haptoglobin bias the extraction unit toward βHb, sterically occluding its access to αHb while still permitting engagement by N1. Kinetic assays show that IsdH actively accelerates hemin release from Hb:Hp. Three-dimensional variability analysis indicates that this likely occurs via a dynamic interface in which receptor motions reposition the extraction unit relative to βHb, collectively supporting a model in which IsdH transiently perturbs the F-helix to promote hemin extraction. Alignment of that model with a previously determined CD163:Hb:Hp structure shows how IsdH may disrupt Hb:Hp recognition by macrophage and monocyte CD163 receptors, helping to explain how it may hinder clearance of Hb:Hp from circulation. In aggregate, these results help define the structural basis for hemin extraction from Hb:Hp and how IsdH may subvert receptor-mediated clearance of the Hb:Hp complex. Significance: Staphylococcus aureus scavenges hemin from host hemoglobin to proliferate, yet most extracellular hemoglobin is sequestered in hemoglobin:haptoglobin (Hb:Hp) complexes, which are rapidly cleared from circulation. To clarify how bacteria access hemin in this context, we now show that the IsdH surface receptor is structurally adapted to extract hemin from Hb:Hp. Our results indicate that IsdH uses distinct NEAT domains to anchor to αHb and selectively extract hemin from βHb. This βHb selectivity is shaped by haptoglobin N-glycans and enhances microbial access to iron. These findings provide a mechanistic framework for targeting heme acquisition as an anti-virulence strategy.

Native mass spectrometry (nMS) is well established for measuring protein masses and stoichiometries using nano-electrospray ionization (nESI), yet salt adduction and source activation energies can limit routine measurements. In this study, we benchmark submicron quartz nanopipette nESI emitters (<50 nm internal diameter) across three mass spectrometry platforms (quadrupole-time-of-flight, quadrupole-Orbitrap, and tribrid-Orbitrap platforms) and a wide protein mass range (17-800 kDa). We analysed holo-myoglobin (17 kDa) over a range of concentrations (10 μM-10 nM) and capillary voltages to determine limits of detection and define a gentle operating regime. We additionally observe reduced Na+ adduction and preservation of the Zn2+ bound metalloproteoform of carbonic anhydrase II (29 kDa). Proteins and protein complexes spanning the mid-to-high mass range including ovalbumin (~44 kDa), malate dehydrogenase (~70 kDa), glutamate dehydrogenase (~350 kDa), β-galactosidase (~465 kDa), and GroEL (~800 kDa), were readily detected using nanopipette emitters. Compared with conventional 1-2 μm internal diameter borosilicate emitters, quartz nanopipettes provided higher signal-to-noise ratios and fewer adducts. Finally, direct analysis of clarified bacterial lysate expressing α-synuclein yielded a clear monomeric charge-state distribution, demonstrating compatibility with complex biological matrices. Collectively, these results establish quartz nanopipette nESI as an instrument-portable, salt-tolerant approach suitable for routine nMS analysis across a broad range of protein molecular weights and sample complexities.

Visual proteomics enables the study of low-abundance proteins and identification of unknown complexes from heterogeneous samples by complementing high-resolution cryogenic electron microscopy (cryoEM) with external inputs on protein identity such as mass spectrometry. Using this approach, we interrogated the exoproteome of the anaerobic cellulose-degrading bacterium Clostridium thermocellum as it carried out biomass degradation. Mass spectrometry indicated a broad exoproteome composition, including cellulose degrading machinery CelA and CipA. A focus on large exoproteome assemblies revealed abundant protein filaments and pleomorphic vesicular structures. Analysis of the most abundant protein filaments yielded an ∼4 □ resolution native structure that, aided by mass spectrometry, de novo modeling, and structural searching, was found to be the aldehyde-alcohol dehydrogenase (AdhE) spirosome. AdhE contained both NAD ⁺ and Fe in their expected binding sites and biochemical and structural analyses of enriched spirosome preparations indicated they were functional. Altered NADH solution concentrations triggered conformational changes in the exoproteomic spirosomes, and the constituent AdhE remained capable of ethanol production. Although the basis for functional extracellular spirosome accumulation in live anaerobic C. thermocellum cultures remains unclear, their abundance in crude exoproteomes suggests their presence could influence biomass fueled C. thermocellum growth.

Iron is an essential micronutrient for nearly all forms of life, including pathogenic microbes that must acquire it from their host during infection. At the host–pathogen interface, humans restrict microbial access to iron through nutritional immunity, which many pathogens overcome by secreting hemophores that scavenge extracellular heme (iron protoporphyrin IX). However, identifying hemophores and other ligand-binding proteins in complex proteomes remains challenging using conventional peptide-based bottom-up mass spectrometry (MS). Here, we introduce ProteoMIX (Proteome Analysis by Mixing), a function-based native top-down proteomics workflow that combines slow-mixing mode native MS with charge reduction to identify ligand-binding proteins directly from complex mixtures. Applying ProteoMIX to the Corynebacterium diphtheriae exoproteome identified ChtA _30–314 , an abundant soluble hemophore generated by proteolytic processing of the surface-exposed ChtA heme receptor. Using native top-down MS sequencing, cell fractionation, and gene deletion, we show that the protease DIP2069 releases ChtA _30–314 by removing ChtA’s transmembrane helix, producing a soluble proteoform that delivers heme to support microbial growth. In contrast, the related paralog ChtC is not processed, enabling C. diphtheriae to generate localization-specific heme-binding proteoforms from related gene products. Extending this approach to Staphylococcus aureus , ProteoMIX also revealed soluble IsdA hemophores that coexist with surface-anchored variants, demonstrating the generality of the method and suggesting that protease-mediated hemophore release may operate across gram-positive pathogens.